Predictive biomarkers

ABSTRACT

The present disclosure relates to the biological markers SAP, SHBG, Myoglobin, MMP-9, and SCF that are predictive for patient response to treatment with a vascular disrupting agent. In particular, the present disclosure relates to biological markers predictive for cancer patient response to treatment with a vascular disrupting agent, as well as methods of treating a cancer patient with a vascular disrupting agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase application of InternationalApplication No. PCT/AU2015/000387, filed Jul. 2, 2015, and claims thepriority of Australian Application No. 2014902541, filed Jul. 2, 2014,the content of both of which is incorporated herein by reference.

FIELD

The present invention relates to biological markers that are predictivefor patient response to treatment with a vascular disrupting agent. Inparticular, the present invention relates to biological markerspredictive for cancer patient response to treatment with a vasculardisrupting agent, as well as methods of treating a cancer patient with avascular disrupting agent.

BACKGROUND

Endothelial cells are highly dependent on tubulin cytoskeleton for theirmotility, invasion, attachment, alignment and proliferation. Vasculardisrupting agents (VDAs), a new class of agents, target endothelialcells and pericytes of the already established tumor vasculature. MostVDAs induce changes in endothelial cell shape by disruption of thecytoskeleton and cell-to-cell junctions. This results in increasedpermeability to proteins and an increased interstitial fluid pressure,which might be sufficient to reduce vessel diameter. Plasma leakage alsoleads to increased blood viscosity resulting in decreased blood flow androuleaux formation. Another factor contributing to the vascular shutdownis the activation of platelets through contact with basement membranecomponents, which are exposed. All together this cascade of eventsresults in vascular shutdown more selectively in tumor endothelium thannormal endothelium. It is suggested that the inhibition of blood flowand the subsequent compromised supply of oxygen and nutrients willinduce necrosis of many tumor cells downstream.

Vascular disrupting agents have been divided into two types, smallmolecule and ligand directed VDAs. Small molecule VDAs are in a moreadvanced stage of clinical development. Small molecule VDAs are eithertubulin-binding agents or flavonoids. Tubulin-binding agents work byacting at the colchicine-binding site of the β-subunit of endothelialtubulin, resulting in depolymerization of microtubules anddisorganization of actin and tubulin (e.g. combretastatin). Disruptionof the endothelial cytoskeleton results in conformational changesleading to loss of blood flow. Tumor-related endothelial cells are muchmore sensitive to the activity of tubulin-binding agents than normalendothelial cells.

Clinical studies investigating the efficacy of VDAs, however, have notbeen able to meet objectives for increases in overall survival or 6month progression free survival across patient populations.

SUMMARY

The present inventors have analysed the level of biological markers inpatients prior to and following infusion of a vascular disrupting agentand found that changes in the pre- to post-administration levels ofcertain biological markers are indicative of the patient responding totreatment with the vascular disrupting agent.

Accordingly, in a first aspect there is provided a method for predictinga response to treatment with a vascular disrupting agent in a patient,the method comprising:

identifying a patient having a level of a biological marker prior toadministration with the vascular disrupting agent which is different tothe level of the biological marker following administration with thevascular disrupting agent,

wherein the biological marker is selected from Serum Amyloid P (SAP),Sex Hormone-Binding Globulin (SHBG), Myoglobin, Matrix Metalloproteinase9 (MMP-9), and/or Stem Cell Factor (SCF), and

wherein a level of a biological marker prior to administration with thevascular disrupting agent that is different to the level of thebiological marker following administration with the vascular disruptingagent is indicative of the patient responding to treatment with thevascular disrupting agent.

In a second aspect there is provided a method of treating cancer in apatient, the method comprising performing the method for predicting aresponse to treatment as described herein and further administering thevascular disrupting agent to the patient.

In one embodiment, the response to treatment with the vasculardisrupting agent is an increase in progression free survival. In oneparticular embodiment, the increase in progression free survival is atleast 10 days, at least 15 days, at least 20 days, at least 30 days, atleast 40 days, or at least 50 days.

In a third aspect there is provided a method of treating cancer in apatient, the method comprising:

(a) identifying a patient having a level of a biological marker prior toadministration with a vascular disrupting agent which is different tothe level of the biological marker following administration with thevascular disrupting agent, and

(b) further administering the vascular disrupting agent to the patient,

wherein the biological marker is selected from SAP, SHBG, Myoglobin,MMP-9, and/or SCF.

In a fourth aspect there is provided a method of treating cancer in apatient, the method comprising administering a vascular disrupting agentto the patient, wherein the patient has a level of a biological markerprior to administration with the vascular disrupting agent which isdifferent to the level of the biological marker following administrationwith the vascular disrupting agent, and wherein the biological marker isselected from SAP, SHBG, Myoglobin, MMP-9, and/or SCF.

In a fifth aspect there is provided a method of selecting a cancerpatient for treatment with a vascular disrupting agent, the methodcomprising performing the method for predicting response to treatment asdescribed herein and selecting a patient having a level of a biologicalmarker prior to administration with the vascular disrupting agent whichis different to the level of the biological marker followingadministration with the vascular disrupting agent, wherein thebiological marker is selected from SAP, SHBG, Myoglobin, MMP-9, and/orSCF.

In one embodiment of first to fifth aspects, the method comprisesdetermining the level of the biological marker in a sample obtained fromthe patient.

While any suitable sample may be used, in one embodiment the sample istumor, blood, serum or plasma.

The skilled person will appreciate that determining the level of abiological marker in a patient sample may comprise determining the levelof a biological marker nucleic acid or a biological marker polypeptide.Thus, in one embodiment, the level of the biological marker isdetermined by measuring the level of biological marker polypeptide.

In one embodiment, the method comprises determining the level of thebiological marker in a sample obtained from the patient prior toadministration with the vascular disrupting agent and/or in a sampleobtained from the patient following administration with the vasculardisrupting agent.

In one embodiment, the biological marker is selected from SAP, SHBG,and/or Myoglobin and the level of the biological marker is lowerfollowing administration with the vascular disrupting agent whencompared to the level of the biological marker prior to administrationwith the vascular disrupting agent.

In one embodiment, the biological marker is SAP and the ratio of thepost administration level to pre-administration level of SAP is ≤1. Inone particular embodiment, the ratio of the post administration level topre-administration level of SAP is ≤0.95.

In another embodiment, the biological marker is SHBG and the ratio ofthe post administration level to pre-administration level of SHBG is ≤1.In one particular embodiment, the ratio of the post administration levelto pre-administration level of SHBG is ≤0.92.

In yet another embodiment, the biological marker is Myoglobin and theratio of the post administration level to pre-administration level ofMyoglobin is ≤1. In one particular embodiment, the ratio of the postadministration level to pre-administration level of Myoglobin is ≤0.93.

In another embodiment, the biological marker is selected from MMP-9and/or SCF and the level of the biological marker is higher followingadministration with the vascular disrupting agent when compared to thelevel of the biological marker prior to administration with the vasculardisrupting agent.

In one embodiment, the biological marker is MMP-9 and the ratio of thepost administration level to pre-administration level of MMP-9 is ≥1. Inone particular embodiment, the ratio of the post administration level topre-administration level of MMP-9 is ≥1.06.

In another embodiment, the biological marker is SCF and the ratio of thepost administration level to pre-administration level of SCF is ≥1.

In yet another embodiment, the vascular disrupting agent is a tubulinpolymerisation inhibitor.

In one particular embodiment, the tubulin polymerisation inhibitor isselected from ABT-751, MPC-6827, AEZS-112, CYT997, MN-029, EPC2407,ZIO-301, vinflunine, vinblastine, vincristine, CA4, Oxi4503, AVE8062,eribulin mesylate, dolastatin, tasidotin, 2-methoxyestradiol, E7974and/or NPI-2358.

In another embodiment, the tubulin polymerisation inhibitor is acompound of formula (I) or a salt, solvate or prodrug thereof

wherein;

X represents O, S, SO, SO₂, Se, SeO, SeO₂ or NR where R is selected fromH, O, optionally substituted acyl, optionally substituted alkenyl,optionally substituted alkyl, optionally substituted aryl, optionallysubstituted cycloalkenyl, optionally substituted cycloalkyl, optionallysubstituted heteroaryl, optionally substituted heterocyclyl, andoptionally substituted sulfonyl;

R^(1A) and R^(1B) each independently represents H, carboxy, cyano,dihalomethoxy, halogen, hydroxy, nitro, pentahaloethyl, phosphorylamino,phosphono, phosphinyl, sulfo, trihaloethenyl, trihalomethanethio,trihalomethoxy, trihalomethyl, optionally substituted acyl, optionallysubstituted acylamino, optionally substituted acylimino, optionallysubstituted acyliminoxy, optionally substituted acyloxy, optionallysubstituted arylalkyl, optionally substituted arylalkoxy, optionallysubstituted alkenyl, optionally substituted alkenyloxy, optionallysubstituted alkoxy, optionally substituted alkyl, optionally substitutedalkynyl, optionally substituted alkynyloxy, optionally substitutedamino, optionally substituted aminoacyl, optionally substitutedaminoacyloxy, optionally substituted aminosulfonyl, optionallysubstituted aminothioacyl, optionally substituted aryl, optionallysubstituted aryloxy, optionally substituted cycloalkenyl, optionallysubstituted cycloalkyl, optionally substituted heteroaryl, optionallysubstituted heterocyclyl, optionally substituted oxyacyl, optionallysubstituted oxyacylamino, optionally substituted oxyacyloxy, optionallysubstituted oxyacylimino, optionally substituted oxysulfinylamino,optionally substituted oxysulfonylamino, optionally substitutedoxythioacyl, optionally substituted oxythioacyloxy, optionallysubstituted sulfinyl, optionally substituted sulfinylamino, optionallysubstituted sulfonyl, optionally substituted sulphonylamino, optionallysubstituted thio, optionally substituted thioacyl, optionallysubstituted thioacylamino, or R^(1A) and R^(1B) together form anoptionally substituted aryl, optionally substituted heterocyclyl,optionally substituted heteroaryl, optionally substituted cycloalkyl, oroptionally substituted cycloalkenyl;

R^(1C) represents C₁₋₃ alkoxy, C₁₋₃ alkylthio, C₁₋₃ alkylamino, or C₁₋₃dialkylamino;

R^(1D) represents hydroxy or amino;

L represents C═O, O, S, SO, SO₂, Se, SeO, SeO₂, C═NZ′, or NR′ where Z′is H, optionally substituted alkyl, optionally substituted aryl oroptionally substituted amino; and where R′ is selected from H, O,optionally substituted acyl, optionally substituted alkenyl, optionallysubstituted alkyl, optionally substituted aryl, optionally substitutedcycloalkenyl, optionally substituted cycloalkyl, optionally substitutedheteroaryl, optionally substituted heterocyclyl, or optionallysubstituted sulfonyl;

R^(2A)-R^(2E) each independently represents H, carboxy, cyano,dihalomethoxy, halogen, hydroxy, nitro, pentahaloethyl, phosphorylamino,phosphono, phosphinyl, sulfo, trihaloethenyl, trihalomethanethio,trihalomethoxy, trihalomethyl, optionally substituted acyl, optionallysubstituted acylamino, optionally substituted acylimino, optionallysubstituted acyliminoxy, optionally substituted acyloxy, optionallysubstituted arylalkyl, optionally substituted arylalkoxy, optionallysubstituted alkenyl, optionally substituted alkenyloxy, optionallysubstituted alkoxy, optionally substituted alkyl, optionally substitutedalkynyl, optionally substituted alkynyloxy, optionally substitutedamino, optionally substituted aminoacyl, optionally substitutedaminoacyloxy, optionally substituted aminosulfonyl, optionallysubstituted aminothioacyl, optionally substituted aryl, optionallysubstituted aryloxy, optionally substituted cycloalkenyl, optionallysubstituted cycloalkyl, optionally substituted heteroaryl, optionallysubstituted heterocyclyl, optionally substituted oxyacyl, optionallysubstituted oxyacylamino, optionally substituted oxyacylimino,optionally substituted oxyacyloxy, optionally substitutedoxysulfinylamino, optionally substituted oxysulfonylamino, optionallysubstituted oxythioacyl, optionally substituted oxythioacyloxy,optionally substituted sulfinyl, optionally substituted sulfinylamino,optionally substituted sulfonyl, optionally substituted sulphonylamino,optionally substituted thio, optionally substituted thioacyl, optionallysubstituted thioacylamino, or optionally substituted thioacyloxy; or anyof R^(2A) and R^(2B), R^(2B) and R^(2C), R^(2C) and R^(2D), and R^(2D)and R^(2E), together form an optionally substituted aryl, optionallysubstituted heterocyclyl, optionally substituted heteroaryl, optionallysubstituted cycloalkyl, or optionally substituted cycloalkenyl; and

Q represents H, CN, halogen, trialkylsilyl, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted acyl, optionally substituted oxyacyl, optionallysubstituted acylamino, optionally substituted aminoacylamino, OR″, SR″or NR″R″, where each R″ independently represents, H, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted aryl, optionally substitutedheteroaryl, optionally substituted heterocyclyl, optionally substitutedacyl and optionally substituted oxyacyl, or NR″′NR″′, where each R″′independently represents H, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted aryl and optionally substituted heteroaryl.

In one embodiment, the compound of formula (I) is a prodrug selectedfrom an ester, an acetate, a phosphate ester or an amide prodrug. Inanother embodiment, the compound of formula (I) is a phosphate prodrug.In a particular embodiment, R^(1D) is hydroxy and the prodrug is aphosphate ester of the hydroxy group. Preferably, the phosphate ester isa disodium phosphate ester.

In another embodiment, the tubulin polymerisation inhibitor is acompound of formula (III) or a salt, solvate or prodrug thereof

In yet another embodiment, the tubulin polymerisation inhibitor isselected from2-methyl-7-hydroxy-3-(3,4,5-trimethoxybenzoyl)-6-methoxybenzofuran(BNC105) and disodium[6-methoxy-2-methyl-3-(3,4,5-trimethoxybenzoyl)-1-benzofuran-7-yl]phosphate(BNC105P).

While the patient may be any patient who would benefit from treatmentwith a vascular disrupting agent, in one embodiment, the patient is arenal cancer patient.

In one embodiment of the second to fifth aspects, the method furthercomprises administering a further therapeutic agent and/or tumorirradiation to the patient.

In one embodiment, the further therapeutic agent is selected from achemotherapeutic, an antibody and/or an immunotherapeutic. In oneparticular embodiment, the therapeutic agent is selected from an mTORinhibitor, tyrosine kinase inhibitor and/or a VEGF inhibitor.

In a sixth aspect there is provided a kit for monitoring the response ofa patient to treatment with a vascular disrupting agent, the kitcomprising agents for detecting and/or quantifying biological markersselected from SAP, SHBG, Myoglobin, MMP-9, and/or SCF.

In one embodiment of the sixth aspect, the agents for detecting and/orquantifying biological markers are attached to a solid support.

In a seventh aspect there is provided a device for directing treatmentof a patient with a vascular disrupting agent, the device comprisesagents for quantifying biological markers selected from SAP, SHBG,Myoglobin, MMP-9, and/or SCF.

In one embodiment, the device comprises a tube, dipstick, array ormulti-well plate.

In one embodiment of the sixth or seventh aspect, the agents fordetecting and/or quantifying biological markers are detectably labelled.

In another embodiment, the agents bind the biological markers.

In yet another embodiment, the agents are antibodies.

As will be apparent, preferred features and characteristics of oneaspect of the invention are applicable to many other aspects of theinvention.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

The invention is hereinafter described by way of the followingnon-limiting Examples and with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Dosing schedule for administration of Afinitor (everolimus) andBNC105P. Samples for the testing of biomarkers were taken at Day 1 (preand post infusion with BNC105P) and at Day 8 (pre and post infusion withBNC105P).

FIG. 2A: Ratio of SAP biomarker level post infusion versus pre infusionwith BNC105P.

FIG. 2B: Survival proportions separated into two groups based on themedian of the ration of[Biomarker]_(POST BNC105P)/[Biomarker]_(BASELINE).

FIG. 3A: Ratio of SHBG biomarker level post infusion versus pre infusionwith BNC105P.

FIG. 3B: Survival proportions separated into two groups based on themedian of the ration of[Biomarker]_(POST BNC105P)/[Biomarker]_(BASELINE).

FIG. 4A: Ratio of Myoglobin biomarker level post infusion versus preinfusion with BNC105P.

FIG. 4B: Survival proportions separated into two groups based on themedian of the ration of[Biomarker]_(POST BNC105P)/[Biomarker]_(BASELINE).

FIG. 5A: Ratio of MMP-9 biomarker level post infusion versus preinfusion with BNC105P.

FIG. 5B: Survival proportions separated into two groups based on themedian of the ration of[Biomarker]_(POST BNC105P)/[Biomarker]_(BASELINE).

FIG. 6A: Ratio of SCF biomarker level post infusion versus pre infusionwith BNC105P.

FIG. 6B: Survival proportions separated into two groups based on themedian of the ration of[Biomarker]_(POST BNC105P)/[Biomarker]_(BASELINE).

KEY TO THE SEQUENCE LISTING

SEQ ID NO:1—Example of amino acid sequence of Serum Amyloid P-Component(SAP)

SEQ ID NO:2—Example of amino acid sequence of Sex Hormone-BindingGlobulin (SHBG)

SEQ ID NO:3—Example of amino acid sequence of Myoglobin (MB)

SEQ ID NO:4—Example of amino acid sequence of Matrix Metalloproteinase-9(MMP-9)

SEQ ID NO:5—Example of amino acid sequence of Stem Cell Factor (SCF)

DETAILED DESCRIPTION General Techniques and Definitions

Unless specifically defined otherwise, all technical and scientificterms used herein shall be taken to have the same meaning as commonlyunderstood by one of ordinary skill in the art (e.g., in moleculargenetics, biochemistry, chemistry and immunology).

Unless otherwise indicated, the molecular genetics, biochemistry, andimmunological techniques utilized in the present invention are standardprocedures, well known to those skilled in the art. Such techniques aredescribed and explained throughout the literature in sources such as, J,Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons(1984), J. Sambrook and Russell., Molecular Cloning: A LaboratoryManual, 3^(rd) edn, Cold Spring Harbour Laboratory Press (2001), R.Scopes, Protein Purification—Principals and Practice, 3^(rd) edn,Springer (1994), T. A. Brown (editor), Essential Molecular Biology: APractical Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover andB. D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4,IRL Press (1995 and 1996), and F. M. Ausubel et al. (editors), CurrentProtocols in Molecular Biology, Greene Pub. Associates andWiley-Interscience (1988, including all updates until present), EdHarlow and David Lane (editors) Antibodies: A Laboratory Manual, ColdSpring Harbour Laboratory, (1988), and J. E. Coligan et al. (editors)Current Protocols in Immunology, John Wiley & Sons (including allupdates until present).

The term “and/or”, e.g., “X and/or Y” shall be understood to mean either“X and Y” or “X or Y” and shall be taken to provide explicit support forboth meanings or for either meaning.

As used herein, the term “about”, unless stated to the contrary, refersto +/−10% of the designated value.

As used herein, the terms “treating”, “treat” or “treatment” includeadministering a vascular disrupting agent to a patient for the purposeof preventing or delaying disease progression and/or to increase theduration of progression free survival as compared to a patient who hasnot been administered the vascular disrupting agent.

As used herein, the terms “response”, “responding”, “response totreatment” or “responding to treatment” refer to a patient having areduction in one or more symptoms or signs of disease and/or a delay orprevention of disease progression, and/or a longer period of diseasefree progression during and/or following treatment with a vasculardisrupting agent when compared to a patient that has not been treatedwith the vascular disrupting agent, and/or to a patient not having achange in the level of a biological marker pre-versuspost-administration with the vascular disrupting agent as describedherein.

“Administering” as used herein is to be construed broadly and includesadministering a composition or therapeutic agent as described herein toa subject or patient as well as providing the composition or therapeuticagent to a cell, such as, for example, by the provision of a prodrug toa patient.

A “sample” may be of any suitable type and may refer, e.g., to amaterial in which the presence or level of biological markers can bedetected. Preferably, the sample is obtained from the subject so thatthe detection of the presence and/or level of biomarkers may beperformed in vitro. Alternatively, the presence and/or level ofbiomarkers can be detected in vivo. The sample can be used as obtaineddirectly from the source or following at least one step of (partial)purification. The sample can be prepared in any convenient medium whichdoes not interfere with the method of the invention. Typically, thesample is an aqueous solution, biological fluid, cells or tissue, suchas tumor tissue. Preferably, the sample is blood, plasma, or serum.Pre-treatment may involve, for example, preparing plasma from blood,diluting viscous fluids, and the like. Methods of treatment can involvefiltration, distillation, separation, concentration, inactivation ofinterfering components, and the addition of reagents. The selection andpre-treatment of biological samples prior to testing is well known inthe art and need not be described further.

Biomarkers Predictive of Patient Response to Treatment

The present inventors have analysed the level of biological markers inpatients prior to and following infusion of a vascular disrupting agent.This led to the finding that patients having a level of a biologicalmarker prior to administration with the vascular disrupting agent whichis different to the level of the biological marker followingadministration with the vascular disrupting agent have an increasedlikelihood of responding to treatment with a vascular disrupting agent,wherein the biological marker is selected from Serum AmyloidP-Component, Sex Hormone-Binding Globulin, Myoglobin, MatrixMetalloproteinase-9, and/or Stem Cell Factor.

Serum Amyloid P-Component (SAP)

Serum Amyloid P-Component (SAP) is the identical serum form of amyloid Pcomponent (AP), a 25 kDa pentameric protein first identified as thepentagonal constituent of in vivo pathological deposits called“amyloid”. SAP is a member of the pentraxins family, characterised bycalcium dependent ligand binding and distinctive flattened 3-jellyrollstructure similar to that of the legume lectins. The cDNA for the Pcomponent of human serum amyloid was isolated and the complete sequenceof the precursor determined in 1985 (Mantzouranis et al., 1985). Asknown in the art, alternative symbols for Serum Amyloid P-Componentinclude Serum Amyloid P, SAP, Short Pentraxin 2, and PTX2 (OMIMReference 104770). The HUGO Gene Nomenclature Committee (HGNC) approvedsymbol is APCS. One non-limiting example of the amino acid sequence ofSAP is provided as SEQ ID NO: 1. The skilled person will appreciate thatthis sequence is merely representative and that some variation in thesequence of SAP exists naturally in the human population.

Sex Hormone-Binding Globulin (SHBG)

Sex Hormone-Binding Globulin is known by the alternative titles:Androgen-Binding Protein, ABP, Testosterone-Binding Beta-Globulin, andTEBG (OMIM Reference 182205). The HGNC approved symbol is SHBG. The SBHGgene was characterised by Gershagen et al. in 1989. One non-limitingexample of the amino acid sequence of SHBG is provided as SEQ ID NO:2.The skilled person will appreciate that this sequence is merelyrepresentative and that some variation in the sequence of SHBG existsnaturally in the human population.

Myoglobin (MB)

Myoglobin (MB) is an iron- and oxygen-binding protein found in themuscle tissue of vertebrates in general and in almost all mammals. It isrelated to hemoglobin, which is the iron- and oxygen-binding protein inblood, specifically in the red blood cells. Another known symbol forMyoglobin is MB, which is also the HGNC approved symbol (OMIM Reference160000). One non-limiting example of the amino acid sequence of MB isprovided as SEQ ID NO:3. The skilled person will appreciate that thissequence is merely representative and that some variation in thesequence of MB exists naturally in the human population.

Matrix Metalloproteinase-9 (MMP-9)

Matrix metalloproteinases are zinc-dependent endopeptidases and belongto a larger family of proteases known as the metzincin superfamily.MMP-9 catalyses the cleavage of gelatin types I and V and collagen typesIV and V. Alternative titles and symbols for MMP-9 include CollagenaseType IV-B, CLG4B, 92-kD Collagenase Type IV, Collagenase Type V, 92-kDGelatinase, Gelatinase B, GELB (OMIM Reference 120361). The HGNCapproved symbol is MMP9. One non-limiting example of the amino acidsequence of MMP-9 is provided as SEQ ID NO:4. The skilled person willappreciate that this sequence is merely representative and that somevariation in the sequence of MMP-9 exists naturally in the humanpopulation.

Stem Cell Factor (SCF)

Stem cell factor (SCF) is a hematopoietic growth factor and ligand forthe KIT tyrosine kinase receptor. Alternative names for SCF include KitLigand, KITLG, KL, KITL, Mast Cell Growth Factor, MGF, MGF Stem CellFactor, Steel Factor, and SF. The HGNC approved Gene Symbol is KITLG(OMIM Reference 184745). One non-limiting example of the amino acidsequence of SCF is provided as SEQ ID NO:5. The skilled person willappreciate that this sequence is merely representative and that somevariation in the sequence of SCF exists naturally in the humanpopulation.

Personalised Medicine

The methods of the present invention can be used to identify thosepatients with an increased likelihood of responding to treatment with avascular disrupting agent. Thus, the terms “prediction” and “predicting”as used herein refer to the likelihood that a patient will respondfavorably to a vascular disrupting agent. In one embodiment, theprediction relates to whether and/or the probability that a patient willhave an increased period of disease free progression followingadministration with a vascular disrupting agent. The predictive methodsof the invention can be used to make treatment decisions by choosing themost appropriate treatment modalities for any particular patient, forexample by directing a particular course of treatment, or adjustingdoses or directing that a treatment be discontinued. For example, themethod of the invention may be used to determine i) whether treatmentwith a vascular disrupting agent in a patient should be commencedfollowing a first test infusion of the agent; ii) whether an alreadycommenced treatment with a vascular disrupting agent should be continuedin view of the patient having an increased likelihood of responding tothe agent; or iii) whether an already commenced treatment with avascular disrupting agent should be ceased in view of the patient havinga reduced likelihood of responding to the agent.

In one embodiment, the patient is an individual likely to benefit fromtreatment with a vascular disrupting agent. By way of non-limitingexamples, patients likely to benefit from treatment with a vasculardisrupting agent include patients with cancer, age related maculardegeneration and endometriosis.

In certain embodiments, the patient that is selected for treatmentaccording to the method of the invention is a cancer patient, inparticular a cancer patient with a solid tumor. A solid tumor can bemalignant or benign. Examples of solid tumors that can be treatedaccording to a method of the present invention include sarcomas andcarcinomas such as, but not limited to: fibrosarcoma, myxosarcoma,liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, colorectal cancer, gastric cancer,pancreatic cancer, breast cancer, ovarian cancer, fallopian tube cancer,primary carcinoma of the peritoneum, prostate cancer, squamous cellcarcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,sebaceous gland carcinoma, papillary carcinoma, papillaryadenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogeniccarcinoma, renal cancer, renal cell carcinoma, hepatoma, livermetastases, bile duct carcinoma, choriocarcinoma, seminoma, embryonalcarcinoma, thyroid carcinoma such as anaplastic thyroid cancer, Wilms'tumor, cervical cancer, testicular tumor, lung carcinoma such as smallcell lung carcinoma and non-small cell lung carcinoma, bladdercarcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, andretinoblastoma.

Biomarker Screening

Any suitable method known to one of skill in the art for detecting thelevel of biological markers in a patient may be used in the presentinvention. Thus, the methods of the invention may involve a degree ofquantification to determine levels of biological markers (also referredto as “biomarkers”) in patient samples. Such quantification is readilyprovided by the inclusion of appropriate control samples or bycomparison to reference data.

In one embodiment, internal controls are included in the methods of thepresent invention. In one embodiment, a preferred internal control isone or more samples taken from one or more individuals who have not beenadministered with a vascular disrupting agent, and/or one or moresamples taken from a patient following administration with a vasculardisrupting agent.

As will be known to those skilled in the art, when internal controls arenot included in each assay conducted, the control may be derived fromreference data (i.e. an established data set). Data pertaining to thecontrol subjects may be selected from the group consisting of:

1. a data set comprising measurements of the level of biomarkers for atypical population of subjects prior to and/or following administrationwith a vascular disrupting agent;

2. a data set comprising measurements of the level of biomarkers for thesubject being tested wherein said measurements have been madepreviously, such as, for example, prior to the administration of avascular disrupting agent or prior to the commencement of treatment;

3. a data set comprising measurements of level of biomarkers for ahealthy individual or a population of healthy individuals; and

4. a data set comprising measurements of the level of biomarkers for anormal individual or a population of normal individuals.

Compounds that bind a biological marker when used according theinvention may be linked to a reagent such as a detectable label to alloweasy detection of binding events in vitro or in vivo. Suitable labelsinclude radioisotopes, dye markers or other imaging reagents fordetection and/or localisation of target molecules. Compounds linked to adetectable label can be used with suitable in vivo imaging technologiessuch as, for example, radiology, fluoroscopy, nuclear magnetic resonanceimaging (MRI), CAT-scanning, positron emission tomography (PET),computerized tomography etc.

In one embodiment, the level of a biological marker polypeptide isdetermined in a patient sample. For example, the method may comprisecontacting a biological sample derived from the patient with a compoundcapable of binding to a biomarker polypeptide, and detecting theformation of complex between the compound and the biomarker polypeptide.The terms “biological marker polypeptide” or “biomarker polypeptide” asused herein include fragments of biomarker polypeptides, including forexample, immunogenic fragments and epitopes of the biomarkerpolypeptide.

In one embodiment, the compound that is used to detect or bind to thebiological marker is an antibody. The term “antibody” as used hereinincludes intact molecules as well as molecules comprising or consistingof fragments thereof, such as, for example Fab, F(ab′)2, Fv and scFv, aswell as engineered variants including diabodies, triabodies, mini-bodiesand single-domain antibodies which are capable of binding an epitopicdeterminant. Thus, antibodies may exist as intact immunoglobulins, or asmodifications in a variety of forms.

Protein detection systems contemplated herein include any known assayfor detecting proteins in a biological sample isolated from a humansubject, such as, for example, SDS/PAGE, isoelectric focussing,2-dimensional gel electrophoresis comprising SDS/PAGE and isoelectricfocussing, an immunoassay, flow cytometry e.g. fluorescence-activatedcell sorting (FACS), a detection based system using an antibody ornon-antibody compound, such as, for example, a small molecule (e.g. achemical compound, agonist, antagonist, allosteric modulator,competitive inhibitor, or non-competitive inhibitor, of the protein). Inaccordance with these embodiments, the antibody or small molecule may beused in any standard solid phase or solution phase assay format amenableto the detection of proteins. Optical or fluorescent detection, such as,for example, using mass spectrometry, MALDI-TOF, biosensor technology,evanescent fiber optics, or fluorescence resonance energy transfer, isclearly encompassed by the present invention. Assay systems suitable foruse in high throughput screening of mass samples, e.g. a high throughputspectroscopy resonance method (e.g. MALDI-TOF, electrospray MS ornano-electrospray MS), are also contemplated. Another suitable proteindetection technique involves the use of Multiple Reaction Monitoring(MRM) in LC-MS (LC/MRM-MS).

Immunoassay formats are also suitable, for example, such as thoseselected from the group consisting of, an immunoblot, a Western blot, adot blot, an enzyme linked immunosorbent assay (ELISA), radioimmunoassay(RIA), enzyme immunoassay. Modified immunoassays utilizing fluorescenceresonance energy transfer (FRET), isotope-coded affinity tags (ICAT),matrix-assisted laser desorption/ionization time of flight (MALDI-TOF),electrospray ionization (ESI), biosensor technology, evanescentfiber-optics technology or protein chip technology are also useful.

In another embodiment, a nucleic acid detection technique is used. Anysuitable technique that allows for the qualitative and/or quantitativeassessment of the level of a biomarker polynucleotide in a sample asknown in the art may be used. The terms “nucleic acid molecule” or“polynucleotide” as used herein refer to an oligonucleotide,polynucleotide or any fragment thereof.

Comparison may be made by reference to a standard control, a controllevel, or reference sample or reference level. For example, levels of atranscribed gene can be determined by Northern blotting, and/or RT-PCR.With the advent of quantitative (real-time) PCR, quantitative analysisof gene expression can be achieved by using appropriate primers for thegene of interest. The nucleic acid may be labelled and hybridised on agene array, in which case the gene concentration will be directlyproportional to the intensity of the radioactive or fluorescent signalgenerated in the array.

Methods for direct sequencing of nucleotide sequences are well known tothose skilled in the art and can be found for example in Ausubel et al.,eds., Short Protocols in Molecular Biology, 3rd ed., Wiley, (1995) andSambrook et al, Molecular Cloning, 3rd ed., Cold Spring HarborLaboratory Press, (2001). Sequencing can be carried out by any suitablemethod, for example, dideoxy sequencing, chemical sequencing orvariations thereof. Direct sequencing has the advantage of determiningvariation in any base pair of a particular sequence.

In one embodiment, the direct sequencing is Next-generationhigh-throughput sequencing. Next-generation sequencing (NGS)technologies include instruments that are capable of sequencing morethan 10¹⁴ kilobase-pairs (kbp) of DNA per instrument run. Sequencingtypically produces a large number of independent reads, eachrepresenting anywhere between 10 to 1000 bases of the nucleic acid.Nucleic acids are generally sequenced redundantly for confidence, withreplicates per unit area being referred to as the coverage (i.e., “10×coverage” or “100× coverage”). Next generation sequencing methods areknown in the art, and are described, for example, in Metzker (2010).

Thus, the terms “Next-generation sequencing” or “NGS” or “NG sequencing”as used herein refer to any sequencing method that determines thenucleotide sequence of either individual nucleic acid molecules (e.g.,in single molecule sequencing) or clonally expanded proxies forindividual nucleic acid molecules in a high through-put fashion (e.g.,greater than 10³, 10⁴, 10⁵ or more molecules are sequencedsimultaneously).

Platforms for next-generation sequencing include, but are not limitedto, Roche/454's Genome Sequencer (GS) FLX System, Illumina/Solexa'sGenome Analyzer (GA), Life/APG's Support Oligonucleotide LigationDetection (SOLiD) system, Polonator's G.007 system, Helicos BioSciences'HeliScope Gene Sequencing system, and Pacific Biosciences' PacBio RSsystem.

Other PCR methods that may be used in carrying out the invention includehybridization based PCR detection systems, TaqMan assay (U.S. Pat. No.5,962,233) and the molecular beacon assay (U.S. Pat. No. 5,925,517).

The nucleic acid may be separated from the sample for testing. Suitablemethods will be known to those of skill in the art. For example, RNA maybe isolated from a sample to be analysed using conventional procedures,such as are supplied by QIAGEN technology. This RNA is thenreverse-transcribed into DNA using reverse transcriptase and the DNAmolecule of interest may then be amplified by PCR techniques usingspecific primers.

Nucleic acid assays may also be performed directly upon patient samples.Hybridisation or amplification assays, such as, for example, Southern orNorthern blot analysis, immunohistochemistry, single-strandedconformational polymorphism analysis (SSCP) and PCR analyses are amongtechniques that are useful in this respect. If desired, target or probenucleic acid may be immobilised to a solid support such as a microtitreplate, membrane, polystyrene bead, glass slide or other solid phase.

In one embodiment, the level of a biological marker may be determinedusing a suitable commercially available test. By way of non-limitingexample, the level of a biomarker may be quantitatively measured usingthe Multi-Analyte Profile (MAP) Technology (Myriad RBM).

The level of the biological marker in a patient sample prior toadministration of the vascular disrupting agent may be determined from asample taken at any time prior to administration of a first dose of thevascular disrupting agent. In one embodiment, the sample is taken within1 month, or within 1 week, or within 3 days, or within 48 hours, orwithin 24 hours, or within 3 hours, or within 1 hour prior to the firstdose of the vascular disrupting agent.

The level of the biological marker in a patient sample followingadministration of the vascular disrupting agent may be determined from asample taken at any time following the administration of the vasculardisrupting agent within which the biological marker is likely tomaintain an altered level due to administration of the agent. In oneembodiment, the sample is taken within 48 hours, or within 24 hours ofadministration with the vascular disrupting agent. In anotherembodiment, the sample is taken within 3 hours of administration withthe vascular disrupting agent. In another embodiment, the sample istaken within 1 hour of administration with the vascular disruptingagent.

As known in the art, the level of a biological marker may be determinedaccording to the detection technique used. Thus, the level of abiological marker may be, for example, a level of expression,transcription or translation of a polynucleotide, the level ofexpression of a polypeptide and/or the concentration of a biologicalmarker in a sample. By way of non-limiting example, the level of abiomarker may be determined or inferred by detection of a label, viacolorimetric change, alterations in signal intensities, such as bydetermining the wavelength or strength of a fluorescent signal, bymeasuring absorbance or optical density, by measuring radioactivesignals. In one embodiment, the level of a biomarker is presented as theconcentration of the biological marker in a sample obtained from thepatient. A concentration of a biological marker may be presented in anysuitable unit such as, for example, ng/ml, μg/ml, mg/ml, pg/μl, or μg/l.

Devices and Kits

The present invention further provides devices, such as predictive ordiagnostic devices and kits for determining the level of a biologicalmarker in a patient or patient sample prior to and/or followingadministration with a vascular disrupting agent. Diagnostic/predictivekits based on the biological markers described above can be developedfor use in predicting an individual's response to treatment with avascular disrupting agent. Such test kits can include devices andinstructions that a subject can use to obtain a sample, e.g., blood,plasma or serum, in some instances with the aid of a health careprovider.

Thus, it will be appreciated that the method of the invention may beused as a “companion diagnostic” to a therapeutic treatment or method inorder to validate or direct the use of the therapeutic. Companiondiagnostics are increasingly finding utility in the justification ofexpensive or high risk treatments which only confer benefit to a subsetof the population. A companion diagnostic test refers to an in vitrodiagnostic device or kit, or an imaging tool, the use of which indicatesan increased likelihood of a patient responding to treatment. In-vitroCompanion Diagnostic tests measure the expression or presence of aspecific biomarker that is linked to a disease condition or therapy.

In one embodiment, the device of the invention, for example a companiondiagnostic device, is an array. The term “array” or “microarray”, asused herein refers to an ordered arrangement of hybridizable arrayelements, such as polynucleotide probes (e.g., oligonucleotides), orbinding reagents (e.g., antibodies), on a substrate. The substrate canbe a solid substrate, such as a glass or silica slide, a bead, a fiberoptic binder, or a semi-solid substrate, such as a nitrocellulosemembrane. The nucleotide sequences can be DNA, RNA, or any permutationsthereof.

In some embodiments, the invention provides compositions and kitscomprising primers and primer pairs, which allow the specificamplification of biomarker polynucleotides, and probes that selectivelyor specifically hybridize to biomarker polynucleotides. Probes may belabeled with a detectable marker, such as, for example, a radioisotope,fluorescent compound, bioluminescent compound, a chemiluminescentcompound, metal chelator or enzyme. Such probes and primers can be usedto detect the presence of polynucleotides in a sample and as a means fordetecting cell expressing proteins encoded by the polynucleotides. Aswill be understood by the skilled artisan, a great many differentprimers and probes may be prepared based on the sequences providedherein and used effectively to amplify, clone and/or determine thepresence and/or levels of the biological markers described herein.

In some embodiments, the device or kit comprises reagents for detectingthe presence of biological marker polypeptides. Such reagents may beantibodies or other binding molecules that specifically bind to apolypeptide. The antibodies or binding molecules may be labeled with adetectable marker, such as, for example, a radioisotope, a fluorescentcompound, a bioluminescent compound, a chemiluminescent compound, ametal chelator, an enzyme, or a particle. Other reagents for performingbinding assays, such as ELISA, may be included in the kit.

In some embodiments, the kits comprise reagents for detecting ordetermining the level of at least two, at least three, at least four, orall of the biological markers described herein. In some embodiments, thekits may further comprise a surface or substrate (such as a microarray)for capture probes for detecting of amplified nucleic acids.

The kits may further comprise a carrier being compartmentalized toreceive in close confinement one or more containers such as vials,tubes, and the like, each of the containers comprising one of theseparate elements to be used in the method. For example, one of thecontainer means may comprise a probe that is or can be detectablylabeled. Such probe may be a polynucleotide or antibody specific for abiomarker. Where the kit utilizes nucleic acid hybridization to detectthe target nucleic acid, the kit may also have containers containingnucleotide(s) for amplification of the target nucleic acid sequenceand/or a container comprising a reporter, such as a biotin-bindingprotein, such as avidin or streptavidin, bound to a reporter molecule,such as an enzymatic, florescent, or radioisotope label. In oneembodiment, one of the containers may comprise an antibody that is orcan be detectably labelled and which binds a biological marker asdescribed herein.

The kit of the invention will typically comprise the container describedabove and one or more other containers comprising materials desirablefrom a commercial and user standpoint, including buffers, diluents,filters, needles, syringes, and package inserts with instructions foruse. A label may be present on the container to indicate that thecomposition is used for a specific purpose and may also indicatedirections for either in vivo or in vitro use, such as those describedabove. The kit can further comprise a set of instructions and materialsfor preparing a tissue or cell sample, for example, blood, plasma orserum, and preparing nucleic acids from the sample.

Vascular Disrupting Agents

Endothelial cells are highly dependent on the tubulin cytoskeleton fortheir motility, invasion, attachment, alignment and proliferation.Vascular disrupting agents (VDAs) target endothelial cells and pericytesof the already established tumor vasculature. Most VDAs induce changesin endothelial cell shape by disruption of the cytoskeleton andcell-to-cell junctions. This results in increased permeability toproteins and an increased interstitial fluid pressure, which might besufficient to reduce vessel diameter. Plasma leakage also leads toincreased blood viscosity resulting in decreased blood flow and rouleauxformation.

Another factor contributing to the vascular shutdown is the activationof platelets through contact with basement membrane components, whichare exposed. All together this cascade of events results in vascularshutdown more selectively in tumor endothelium than normal endothelium.As stated previously, it is suggested that the inhibition of blood flowand the subsequent compromised supply of oxygen and nutrients willinduce necrosis of many tumor cells downstream.

Vascular disrupting agents have been divided into two types, smallmolecule and ligand directed VDAs. Small molecule VDAs are in a moreadvanced stage of clinical development. Small molecule VDAs includetubulin-binding agents and flavonoids. Tubulin-binding agents areproposed to act at the colchicine-binding site of the β-subunit ofendothelial cell tubulin, resulting in depolymerization of microtubulesand disorganization of actin and tubulin (e.g. CA4 (combretastatin)).

Disruption of the endothelial cytoskeleton results in cell morphologychanges leading to reduction or cessation of blood flow. Tumor-relatedendothelial cells are much more sensitive to the activity oftubulin-binding agents than normal endothelial cells. ASA404 is asmall-molecule flavonoid VDA with activity involving inhibition ofpathways that up regulate the nuclear transcription factor NfκB andproduction of TNF-α and other cytokines.

Thus, in one embodiment, the vascular disrupting agent is a TubulinPolymerization Inhibitor (TPI). As used herein the term “tubulinpolymerisation inhibitor” refers to any and all compounds or moleculeswhich directly interact with tubulin and inhibit tubulin polymerisationand/or depolymerise tubulin and as a consequence interferes with thephysiological function of microtubules. Tubulin polymerisationinhibitors (TPIs) are also referred to as microtubule “destabilizing”agents. Such compounds should be contrasted with tubulin interactingcompounds like taxanes and epothilones which stabilise tubulin polymersand inhibit tubulin depolymerisation (i.e., microtubule stabilisingagents).

Microtubules are filamentous polymers that are key components of thecell cytoskeleton. They are dynamic structures fluctuating betweenstates of polymerisation and depolymerisation. This property enablesmicrotubules to modulate cell shape, adhesion, migration andproliferation. TPIs interfere with microtubule integrity, leading tocytoskeletal changes of the endothelial cells that line the bloodvessels of the tumour. As a result, these usually flat cells become morerounded, and lose their cell to cell contact. These events lead tonarrowing of tumour blood vessels and ultimately occlusion of blood flowthrough the vessels. TPIs directly disrupt microtubule polymerisationprocesses and consequently have the ability to effect cell shape changesand inhibit cell proliferation. These properties are central to the useof TPIs as therapeutics for the treatment of cancer and in the method ofthe present invention.

TPIs may also be classified based on their specific tubulin bindingsite. Binding of vinca alkaloids to tubulin defines a site that mediatesthe tubulin destabilization activity seen with these compounds. The“vinca” site has been shown to directly bind a number of compounds thateffect destabilization of tubulin. Examples of TPI's that bind to thevinca site include vinflunine, vinblastine, vincristine, vinorelbine,dolastatin, tasidotin and E7974.

Colchicine binding to tubulin defines an independent binding site thatlike in the case of the “vinca” site causes destabilization of tubulin.Although TPI's binding to the “vinca” sites have been successful asanti-cancer chemotherapeutics, “colchicine” site binders have been incomparison neglected, possibly due to the lack of therapeutic marginsoffered by colchicine. However, more recently a number of “colchicine”site binding agents have been described that have the ability to causedisruption of blood vessels within solid tumors. Many of the“colchicine” site binding agents are based on natural products such ascombretastatins (CA4P, OXi-4503, AVE-8062), colchicines (ZD6126) andphenylahistin (NPI-2358) while others are small molecules which bind tothe colchicine site (ABT-751, MPC-6827, AEZS-112, CYT-997, MN-029,EPC2407, ZIO-301, 2ME2, ZD6126 and NPI-2358).

TPI compounds are important in the treatment of cancers primarily as aresult of their capacity to selectively shut down blood flow through atumour. Targeting tubulin polymerisation inhibition has been a very wellvalidated anti-cancer approach through the development and now extensiveclinical use of chemotherapeutic TPIs.

Examples of TPIs suitable for use in the present invention includeABT-751 (E7010, Abbott), MPC-6827 (Azixa™, Myriad Pharmaceuticals),AEZS-112 (ZEN-012, Eterna Zentaris), CYT997 (Cytopia), MN-029(Denibulin, MediciNova/Angiogene), EPC2407 (EpiCept), ZIO-301(Indibulin, Ziopharm Oncology), Vinflunine (Javlor, Pierre FabreMedicament) as well as other vinca alkaloids (e.g., vinblastin,vincristine, and vinorelbine), combretastatins (CA4 (Zybrestat™,OXiGENE), Oxi4503 (OXiGENE), and AVE8062 (AC7700, Sanofi Aventis)),Eribulin Mesylate (E7389, Eisai), Dolastatin 10 (NCI), Tasidotin(synthadotin, Genzyme), 2-methoxyestradiol (2ME2 or Panzem®, EntreMed),E7974 (Eisai), and NPI-2358 (Nereus Pharmaceuticals). Examples of TPIstructures are provided in Table 1.

TABLE 1 Examples of TPI structures

In an embodiment the TPI is selected from a compound of formula (I) orsalts, solvates or prodrugs thereof

wherein;

X represents O, S, SO, SO₂, Se, SeO, SeO₂ or NR where R is selected fromH, O, optionally substituted acyl, optionally substituted alkenyl,optionally substituted alkyl, optionally substituted aryl, optionallysubstituted cycloalkenyl, optionally substituted cycloalkyl, optionallysubstituted heteroaryl, optionally substituted heterocyclyl, andoptionally substituted sulfonyl;

R^(1A) and R^(1B) each independently represents H, carboxy, cyano,dihalomethoxy, halogen, hydroxy, nitro, pentahaloethyl, phosphorylamino,phosphono, phosphinyl, sulfo, trihaloethenyl, trihalomethanethio,trihalomethoxy, trihalomethyl, optionally substituted acyl, optionallysubstituted acylamino, optionally substituted acylimino, optionallysubstituted acyliminoxy, optionally substituted acyloxy, optionallysubstituted arylalkyl, optionally substituted arylalkoxy, optionallysubstituted alkenyl, optionally substituted alkenyloxy, optionallysubstituted alkoxy, optionally substituted alkyl, optionally substitutedalkynyl, optionally substituted alkynyloxy, optionally substitutedamino, optionally substituted aminoacyl, optionally substitutedaminoacyloxy, optionally substituted aminosulfonyl, optionallysubstituted aminothioacyl, optionally substituted aryl, optionallysubstituted aryloxy, optionally substituted cycloalkenyl, optionallysubstituted cycloalkyl, optionally substituted heteroaryl, optionallysubstituted heterocyclyl, optionally substituted oxyacyl, optionallysubstituted oxyacylamino, optionally substituted oxyacyloxy, optionallysubstituted oxyacylimino, optionally substituted oxysulfinylamino,optionally substituted oxysulfonylamino, optionally substitutedoxythioacyl, optionally substituted oxythioacyloxy, optionallysubstituted sulfinyl, optionally substituted sulfinylamino, optionallysubstituted sulfonyl, optionally substituted sulphonylamino, optionallysubstituted thio, optionally substituted thioacyl, optionallysubstituted thioacylamino, or R^(1A) and R^(1B) together form anoptionally substituted aryl, optionally substituted heterocyclyl,optionally substituted heteroaryl, optionally substituted cycloalkyl, oroptionally substituted cycloalkenyl;

R^(1C) represents C₁₋₃ alkoxy, C₁₋₃ alkylthio, C₁₋₃ alkylamino, or C₁₋₃dialkylamino;

R^(1D) represents hydroxy or amino;

L represents C═O, O, S, SO, SO₂, Se, SeO, SeO₂, C═NZ′, or NR′ where Z′is H, optionally substituted alkyl, optionally substituted aryl oroptionally substituted amino; and where R′ is selected from H, O,optionally substituted acyl, optionally substituted alkenyl, optionallysubstituted alkyl, optionally substituted aryl, optionally substitutedcycloalkenyl, optionally substituted cycloalkyl, optionally substitutedheteroaryl, optionally substituted heterocyclyl, or optionallysubstituted sulfonyl;

R^(2A)-R^(2E) each independently represents H, carboxy, cyano,dihalomethoxy, halogen, hydroxy, nitro, pentahaloethyl, phosphorylamino,phosphono, phosphinyl, sulfo, trihaloethenyl, trihalomethanethio,trihalomethoxy, trihalomethyl, optionally substituted acyl, optionallysubstituted acylamino, optionally substituted acylimino, optionallysubstituted acyliminoxy, optionally substituted acyloxy, optionallysubstituted arylalkyl, optionally substituted arylalkoxy, optionallysubstituted alkenyl, optionally substituted alkenyloxy, optionallysubstituted alkoxy, optionally substituted alkyl, optionally substitutedalkynyl, optionally substituted alkynyloxy, optionally substitutedamino, optionally substituted aminoacyl, optionally substitutedaminoacyloxy, optionally substituted aminosulfonyl, optionallysubstituted aminothioacyl, optionally substituted aryl, optionallysubstituted aryloxy, optionally substituted cycloalkenyl, optionallysubstituted cycloalkyl, optionally substituted heteroaryl, optionallysubstituted heterocyclyl, optionally substituted oxyacyl, optionallysubstituted oxyacylamino, optionally substituted oxyacylimino,optionally substituted oxyacyloxy, optionally substitutedoxysulfinylamino, optionally substituted oxysulfonylamino, optionallysubstituted oxythioacyl, optionally substituted oxythioacyloxy,optionally substituted sulfinyl, optionally substituted sulfinylamino,optionally substituted sulfonyl, optionally substituted sulphonylamino,optionally substituted thio, optionally substituted thioacyl, optionallysubstituted thioacylamino, or optionally substituted thioacyloxy; or anyof R^(2A) and R^(2B), R^(2B) and R^(2C), R^(2C) and R^(2D), and R^(2D)and R^(2E), together form an optionally substituted aryl, optionallysubstituted heterocyclyl, optionally substituted heteroaryl, optionallysubstituted cycloalkyl, or optionally substituted cycloalkenyl; and

Q represents H, CN, halogen, trialkylsilyl, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted acyl, optionally substituted oxyacyl, optionallysubstituted acylamino, optionally substituted aminoacylamino, OR″, SR″or NR″R″, where each R″ independently represents, H, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted aryl, optionally substitutedheteroaryl, optionally substituted heterocyclyl, optionally substitutedacyl and optionally substituted oxyacyl, or NR″′NR″′, where each R″′independently represents H, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted aryl and optionally substituted heteroaryl.

In some embodiments X is selected from

O,

S,

SO,

SO₂,

Se,

SeO,

SeO₂ or

NR where R is selected from

H,

O,

optionally substituted acyl selected from H—C(O)—, C₁-C₁₀ alkyl-C(O)—(preferably C₁-C₆ alkyl, more preferably C₁-C₃ alkyl), C₄-C₈cycloalkyl-C(O)—, C₆-C₁₄ aryl-C(O)—, heteroaryl-C(O)— having from 2 to10 carbon atoms and 1 to 4 heteroatoms selected from oxygen, nitrogen,selenium, and sulfur (including oxides of sulfur, selenium and nitrogen)within the ring or heterocyclyl-C(O)— having from 1 to 8 carbon atomsand from 1 to 4 heteroatoms selected from nitrogen, sulfur, oxygen,selenium or phosphorous within the ring. Examples of suitable acylgroups include formyl acetyl, propionyl, benzoyl (optionally substitutedwith methyl, methoxy, halogen, nitro, trifluoromethyl or cyano);

optionally substituted monovalent C₂-C₁₀ alkenyl group which may bestraight chained or branched (preferably C₂-C₆ alkenyl) having at least1 or from 1-2 carbon to carbon double bonds. Examples of suitableoptionally substituted alkenyl groups include, ethenyl, n-propenyl,iso-propenyl, but-2-enyl, 1-propenyl, vinyl, nitrovinyl, cyano vinyl, ortrifluorovinyl and styryl (optionally substituted with methyl, methoxy,halogen, nitro, trifluoromethane or cyano);

optionally substituted C₁-C₁₀ alkyl (preferably C₁-C₆ alkyl, morepreferably C₁-C₃ alkyl). Examples of suitable alkyl groups includemethyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, n-hexyl,1-hydroxyethyl, 1-thioethyl, methoxyiminomethyl, ethoxyiminomethyl,1-(hydroxyimino)ethyl, 1-(hydroxyimino)propyl, 1-hydrazinoethyl,1-hydrazinopropyl, hydroxyiminomethyl, 2-oxopropyl, 2-oxobutyl,3-oxobutyl, 3-oxopentyl, nitromethyl, 1-nitromethyl, and 2-nitroethyl;

optionally substituted C₆-C₁₄ aryl;

optionally substituted C₄-C₈ cycloalkenyl;

optionally substituted C₃-C₈ cycloalkyl;

optionally substituted heteroaryl having from 2 to 10 carbon atoms and 1to 4 heteroatoms selected from oxygen, nitrogen, selenium, and sulfur(including oxides of sulfur, selenium and nitrogen) within the ring;

optionally substituted heterocyclyl having from 1 to 8 carbon atoms andfrom 1 to 4 heteroatoms selected from nitrogen, sulfur, oxygen, seleniumor phosphorous within the ring; and optionally substituted sulfonylselected from H—S(O)₂—, C₁-C₁₀ alkyl-S(O)₂— (preferably C₁-C₆ alkyl,more preferably C₁-C₃ alkyl), C₃-C₈ cycloalkyl-S(O)₂—, C₆-C₁₄aryl-S(O)₂—, heteroaryl-S(O)₂— where the heteroaryl group has from 2 to10 carbon atoms and 1 to 4 heteroatoms selected from oxygen, nitrogen,selenium, and sulfur (including oxides of sulfur, selenium and nitrogen)within the ring, and heterocyclyl-S(O)₂— where the heterocyclyl grouphas from 1 to 8 carbon atoms and from 1 to 4 heteroatoms selected fromnitrogen, sulfur, oxygen, selenium or phosphorous within the ring.Examples of sulfonyl groups include methylsulfonyl, ethylsulfonyl,benzenesulfonyl (optionally substituted with methyl, methoxy, halogen,nitro, trifluoromethane or cyano), methoxycarbo, trifluoromethane;

In some embodiments R^(1A)-R^(1B) and R^(2A)-R^(2E) are independentlyselected from the following groups:

hydrogen;

C₁-C₁₀ alkyl, preferably C₁-C₆ alkyl, more preferably C₁-C₃ alkyl.Examples of suitable alkyl groups include methyl, ethyl, n-propyl,iso-propyl, n-butyl, iso-butyl and n-hexyl;

substituted C₁-C₁₀ alkyl group, preferably C₁-C₆ alkyl, more preferablyC₁-C₃ alkyl. Examples of substituted alkyl groups include1-hydroxyethyl, 1-thioethyl, methoxyiminomethyl, ethoxyiminomethyl,1-(hydroxyimino)ethyl, 1-(hydroxyimino)propyl, 1-hydrazinoethyl,1-hydrazinopropyl, hydroxyiminomethyl, 2-oxopropyl, 2-oxobutyl,3-oxobutyl, 3-oxopentyl, nitromethyl, 1-nitromethyl, and 2-nitroethyl;

optionally substituted acyl group selected from H—C(O)—, C₁-C₁₀alkyl-C(O)— (preferably C₁-C₆ alkyl, more preferably C₁-C₃ alkyl), C₃-C₈cycloalkyl-C(O)—, C₆-C₁₄ aryl-C(O)—, heteroaryl-C(O)— where theheteroaryl group has from 2 to 10 carbon atoms and 1 to 4 heteroatomsselected from oxygen, nitrogen, selenium, and sulfur (including oxidesof sulfur, selenium and nitrogen) within the ring) andheterocyclyl-C(O)— where the heterocyclyl group has from 1 to 8 carbonatoms and from 1 to 4 heteroatoms selected from nitrogen, sulfur,oxygen, selenium or phosphorous within the ring). Examples of acylgroups include formyl acetyl, propionyl, benzoyl (optionally substitutedwith methyl, methoxy, halogen, nitro, trifluoromethyl or cyano);

optionally substituted C₁-C₁₀ alkoxy group, preferably C₁-C₆ alkoxy,more preferably C₁-C₃ alkoxy. Examples of suitable alkoxy groups includemethoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, tert-butoxy,sec-butoxy, n-pentoxy, n-hexoxy and 1,2-dimethylbutoxy; optionallysubstituted oxyacyl group selected from HOC(O)—, C₁-C₁₀ alkyl-OC(O)—(preferably preferably C₁-C₆ alkyl, more preferably C₁-C₃ alkyl), C₃-C₈cycloalkyl-OC(O)—, C₆-C₁₄ aryl-OC(O)—, heteroaryl-OC(O)— where theheteroaryl group has from 2 to 10 carbon atoms and 1 to 4 heteroatomsselected from oxygen, nitrogen, selenium, and sulfur (including oxidesof sulfur, selenium and nitrogen) within the ring, andheterocyclyl-OC(O)— where the heterocyclyl group has from 1 to 8 carbonatoms and from 1 to 4 heteroatoms selected from nitrogen, sulfur,oxygen, selenium or phosphorous within the ring. Examples of oxyacylgroups include methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl,butyloxycarbonyl, isobutyloxycarbonyl;

optionally substituted acyloxy group selected from —OC(O)—(C₁-C₁₀ alkyl)(preferably C₁-C₆ alkyl, more preferably C₁-C₃ alkyl), —OC(O)—(C₆-C₁₄aryl), —C(O)O-heteroaryl where the heteroaryl group has from 2 to 10carbon atoms and 1 to 4 heteroatoms selected from oxygen, nitrogen,selenium, and sulfur (including oxides of sulfur, selenium and nitrogen)within the ring, and —C(O)O-heterocyclyl where the heterocyclyl grouphas from 1 to 8 carbon atoms and from 1 to 4 heteroatoms selected fromnitrogen, sulfur, oxygen, selenium or phosphorous within the ring.Examples of acyloxy groups include acetoxy and propoxy;

optionally substituted (C₆-C₁₄ aryl)-(C₁-C₁₀ alkyl) group. Preferablythe aryl group is C₆-C₁₀ aryl. Preferably the alkyl group is C₁-C₆alkyl, more preferably C₁-C₃ alkyl. Examples of substituted arylalkylgroups include benzyl, phenethyl, 1-hydroxybenzyl, and 1-thiobenzyl;

optionally substituted sulfinyl group selected from H—S(O)—, C₁-C₁₀alkyl-S(O)— (preferably C₁-C₆ alkyl, more preferably C₁-C₃ alkyl), C₃-C₈cycloalkyl-S(O)—, C₆-C₁₄ aryl-S(O)— (preferably, the aryl group has from6 to 14 carbon atoms), heteroaryl-S(O)— where the heteroaryl group hasfrom 2 to 10 carbon atoms and 1 to 4 heteroatoms selected from oxygen,nitrogen, selenium, and sulfur (including oxides of sulfur, selenium andnitrogen) within the ring, and heterocyclyl-S(O)— where the heterocyclylgroup has from 1 to 8 carbon atoms and from 1 to 4 heteroatoms selectedfrom nitrogen, sulfur, oxygen, selenium or phosphorous within the ring.Examples of sulfinyl groups include methylsulfinyl, ethylsulfinyl,benzene sulfinyl (optionally substituted with methyl, methoxy, halogen,nitro, trifluoromethane or cyano), methoxysulfinyl, ethoxysulfinyl;

optionally substituted sulfonyl group selected from H—S(O)₂—, C₁-C₁₀alkyl-S(O)₂— (preferably C₁-C₆ alkyl, more preferably C₁-C₃ alkyl),C₃-C₈ cycloalkyl-S(O)₂—, C₆-C₁₄ aryl-S(O)₂—, heteroaryl-S(O)₂— where theheteroaryl group has from 2 to 10 carbon atoms and 1 to 4 heteroatomsselected from oxygen, nitrogen, selenium, and sulfur (including oxidesof sulfur, selenium and nitrogen) within the ring, andheterocyclyl-S(O)₂— where the heterocyclyl group has from 1 to 8 carbonatoms and from 1 to 4 heteroatoms selected from nitrogen, sulfur,oxygen, selenium or phosphorous within the ring. Examples of sulfonylgroups include methylsulfonyl, ethylsulfonyl, benzenesulfonyl(optionally substituted with methyl, methoxy, halogen, nitro,trifluoromethane or cyano), methoxycarbo, trifluoromethane;

optionally substituted oxyacylamino group of the formula —NR*C(O)OR*where each R* is independently hydrogen, C₁-C₁₀ alkyl (preferably C₁-C₆alkyl, more preferably C₁-C₃ alkyl), C₃-C₈ cycloalkyl, C₆-C₁₄ aryl,heteroaryl having from 2 to 10 carbon atoms and 1 to 4 heteroatomsselected from oxygen, nitrogen, selenium, and sulfur (including oxidesof sulfur, selenium and nitrogen) within the ring and heterocyclylhaving from 1 to 8 carbon atoms and from 1 to 4 heteroatoms selectedfrom nitrogen, sulfur, oxygen, selenium or phosphorous within the ring.Examples of oxyacylamino groups include methoxycarbonylamido, andethoxycarbonyl amido;

optionally substituted oxythioacyl group selected from HO—C(S)—, C₁-C₁₀alkylO—C(S)— (preferably C₁-C₆ alkyl, more preferably C₁-C₃ alkyl),C₃-C₈ cycloalkylO—C(S)—, C₆-C₁₄ arylO—C(S)—, heteroarylO—C(S)— where theheteroaryl group has from 2 to 10 carbon atoms and 1 to 4 heteroatomsselected from oxygen, nitrogen, selenium, and sulfur (including oxidesof sulfur, selenium and nitrogen) within the ring, andheterocyclylO—C(S)— where the heterocyclyl group has from 1 to 8 carbonatoms and from 1 to 4 heteroatoms selected from nitrogen, sulfur,oxygen, selenium or phosphorous within the ring. Examples of oxythioacylgroups include methoxythiocarbonyl and ethoxythiocarbonyl;

optionally substituted thioacyloxy group selected from H—C(S)—O—, C₁-C₁₀alkyl-C(S)—O— (preferably C₁-C₆ alkyl, more preferably C₁-C₃ alkyl),C₃-C₈ cycloalkyl-C(S)—O—, C₆-C₁₄ aryl-C(S)—O—, heteroaryl-C(S)—O— wherethe heteroaryl group has from 2 to 10 carbon atoms and 1 to 4heteroatoms selected from oxygen, nitrogen, selenium, and sulfur(including oxides of sulfur, selenium and nitrogen) within the ring, andheterocyclyl-C(S)—O— where the heterocyclyl group has from 1 to 8 carbonatoms and from 1 to 4 heteroatoms selected from nitrogen, sulfur,oxygen, selenium or phosphorous within the ring. Examples of thioacyloxygroups include thionoacetoxy and thionopropionoxy;

optionally substituted sulfinylamino group selected from H—S(O)—NR*—,C₁-C₁₀ alkyl-S(O)—NR*— (preferably the alkyl groups are C₁-C₆ alkyl,more preferably C₁-C₃ alkyl), C₃-C₈ cycloalkyl-S(O)—NR*—, C₆-C₁₄aryl-S(O)—NR*—, heteroaryl-S(O)—NR*— where the heteroaryl group has from2 to 10 carbon atoms and 1 to 4 heteroatoms selected from oxygen,nitrogen, selenium, and sulfur (including oxides of sulfur, selenium andnitrogen) within the ring, and heterocyclyl-S(O)—NR*— where theheterocyclyl group has from 1 to 8 carbon atoms and from 1 to 4heteroatoms selected from nitrogen, sulfur, oxygen, selenium orphosphorous within the ring. R* is independently hydrogen, C₁-C₁₀ alkyl(preferably C₁-C₆ alkyl, more preferably C₁-C₃ alkyl), C₃-C₈cycloalkyl,C₆-C₁₄ aryl, heteroaryl having from 2 to 10 carbon atoms and 1 to 4heteroatoms selected from oxygen, nitrogen, selenium, and sulfur(including oxides of sulfur, selenium and nitrogen) within the ring, andheterocyclyl having from 1 to 8 carbon atoms and from 1 to 4 heteroatomsselected from nitrogen, sulfur, oxygen, selenium or phosphorous withinthe ring. Examples of sulfinylamino groups include methylsulfinylamino,ethylsulfinylamino, and benzenesulfinylamino (optionally substitutedwith methyl, methoxy, halogen, nitro, trifluoromethane or cyano);

amino group;

substituted amino groups of the formula —NR*R* where each R* isindependently hydrogen, C₁-C₁₀ alkyl (preferably C₁-C₆ alkyl, morepreferably C₁-C₃ alkyl), C₃-C₈ cycloalkyl, C₆-C₁₄ aryl, heteroarylhaving from 2 to 10 carbon atoms and 1 to 4 heteroatoms selected fromoxygen, nitrogen, selenium, and sulfur (including oxides of sulfur,selenium and nitrogen) within the ring and heterocyclyl having from 1 to8 carbon atoms and from 1 to 4 heteroatoms selected from nitrogen,sulfur, oxygen, selenium or phosphorous within the ring. Examples ofsubstituted amino groups include residues of L-valine, D-valine,L-alanine, D-alanine, aspartic acid, and alanylserine, N-methylamino,and N,N′-dimethylamino;

optionally substituted sulfonylamino group selected from H—S(O)₂—NR*—,C₁-C₁₀ alkyl-S(O)₂—NR*— (preferably C₁-C₆ alkyl, more preferably C₁-C₃alkyl), C₃-C₈ cycloalkyl-S(O)₂—NR*—, C₆-C₁₄ aryl-S(O)₂—NR*—,heteroaryl-S(O)₂—NR*— where the heteroaryl group has from 2 to 10 carbonatoms and 1 to 4 heteroatoms selected from oxygen, nitrogen, selenium,and sulfur (including oxides of sulfur, selenium and nitrogen) withinthe ring, and heterocyclyl-S(O)₂-NR*— where the heterocyclyl group hasfrom 1 to 8 carbon atoms and from 1 to 4 heteroatoms selected fromnitrogen, sulfur, oxygen, selenium or phosphorous within the ring. R* isindependently hydrogen, C₁-C₁₀ alkyl (preferably C₁-C₆ alkyl, morepreferably C₁-C₃ alkyl), C₃-C₈ cycloalkyl, C₆-C₁₄ aryl, heteroarylhaving from 2 to 10 carbon atoms and 1 to 4 heteroatoms selected fromoxygen, nitrogen, selenium, and sulfur (including oxides of sulfur,selenium and nitrogen) within the ring and heterocyclyl having from 1 to8 carbon atoms and from 1 to 4 heteroatoms selected from nitrogen,sulfur, oxygen, selenium or phosphorous within the ring. Examples ofsulfonylamino groups include methylsulfonylamino, ethylsulfonylamino andbenzene sulfonylamino (optionally substituted with methyl, methoxy,halogen, nitro, trifluoromethane or cyano);

optionally substituted oxysulfinylamino group selected fromHO—S(O)—NR*—, C₁-C₁₀ alkylO—S(O)—NR*— (preferably C₁-C₆ alkyl, morepreferably C₁-C₃ alkyl), C₃-C₈ cycloalkylO—S(O)—NR*—, C₆-C₁₄arylO—S(O)—NR*—, heteroarylO—S(O)—NR*— where the heteroaryl group hasfrom 2 to 10 carbon atoms and 1 to 4 heteroatoms selected from oxygen,nitrogen, selenium, and sulfur (including oxides of sulfur, selenium andnitrogen) within the ring, and heterocyclylO—S(O)—NR*— where theheterocyclyl group has from 1 to 8 carbon atoms and from 1 to 4heteroatoms selected from nitrogen, sulfur, oxygen, selenium orphosphorous within the ring. R* is independently hydrogen, C₁-C₁₀ alkyl(preferably C₁-C₆ alkyl, more preferably C₁-C₃ alkyl), C₃-C₈ cycloalkyl,C₆-C₁₄ aryl, heteroaryl having from 2 to 10 carbon atoms and 1 to 4heteroatoms selected from oxygen, nitrogen, selenium, and sulfur(including oxides of sulfur, selenium and nitrogen) within the ring andheterocyclyl having from 1 to 8 carbon atoms and from 1 to 4 heteroatomsselected from nitrogen, sulfur, oxygen, selenium or phosphorous withinthe ring. Examples of suitable oxysulfinylamino groups includemethoxysulfinylamino and ethoxysulfinylamino;

optionally substituted oxysulfonylamino group selected fromHO—S(O)₂—NR*—, C₁-C₁₀ alkylO—S(O)₂—NR*— (preferably C₁-C₆ alkyl, morepreferably C₁-C₃ alkyl), C₃-C₈ cycloalkylO—S(O)₂—NR*—, C₆-C₁₄arylO—S(O)₂—NR*—, heteroarylO—S(O)₂—NR*— where the heteroaryl group hasfrom 2 to 10 carbon atoms and 1 to 4 heteroatoms selected from oxygen,nitrogen, selenium, and sulfur (including oxides of sulfur, selenium andnitrogen) within the ring, and heterocyclylO—S(O)₂—NR*— where theheterocyclyl group has from 1 to 8 carbon atoms and from 1 to 4heteroatoms selected from nitrogen, sulfur, oxygen, selenium orphosphorous within the ring. R* is independently hydrogen, C₁-C₁₀ alkyl(preferably C₁-C₆ alkyl, more preferably C₁-C₃ alkyl), C₃-C₈ cycloalkyl,C₆-C₁₄ aryl, heteroaryl having from 2 to 10 carbon atoms and 1 to 4heteroatoms selected from oxygen, nitrogen, selenium, and sulfur(including oxides of sulfur, selenium and nitrogen) within the ring andheterocyclyl having from 1 to 8 carbon atoms and from 1 to 4 heteroatomsselected from nitrogen, sulfur, oxygen, selenium or phosphorous withinthe ring. Examples of oxysulfonylamino groups includemethoxysulfonylamino and ethoxysulfonylamino;

optionally substituted C₂-C₁₀ alkenyl group which may be straightchained or branched and have at least 1 or from 1-2 carbon to carbondouble bonds. Preferably optionally substituted C₂-C₆ alkenyl. Examplesof suitable optionally substituted alkenyl groups include ethenyl,n-propenyl, iso-propenyl, but-2-enyl, 1-propenyl, vinyl, nitrovinyl,cyano vinyl, or trifluorovinyl and styryl (optionally substituted withmethyl, methoxy, halogen, nitro, trifluoromethane or cyano);

optionally substituted C₂-C₁₀ alkynyl group having at least 1 or from1-2 carbon to carbon triple bonds. Preferably C₂-C₆ alkynyl. Examples ofsuitable alkynyl groups include 1-propynyl, ethynyl, propargyl,pent-2-ynyl and trimethylsilylethynyl.

In some embodiments R^(1C) is selected from the following groups:

C₁₋₃ alkoxy. Examples of suitable alkoxy groups include methoxy, ethoxy,n-propoxy, and iso-propoxy;

C₁₋₃ alkylthio. Examples of suitable alkylthio groups include methyl-S—,ethyl-S—, 1-thio-propyl, 2-thio-propyl and iso-propyl-S—;

C₁₋₃ alkylamino. Examples of suitable alkylamino groups includemethylamino, ethylamino, 1-amino-propyl, 2-amino-propyl, andiso-propyl-amino; and

C₁₋₃ dialkylamino. Examples of suitable alkylamino groups includedimethylamino, diethylamino, dipropylamino, ethylmethylamino,propylmethylamino, and propylmethylamino, where the alkyl groups may bestraight chained or branched.

In some embodiments R^(1D) is selected from a hydroxy group and an aminogroup.

In some embodiments L is selected from the following groups:

C═O,

O,

S,

SO,

SO₂,

Se,

SeO,

SeO₂,

C═NZ′ where Z′ is H, optionally substituted C₁-C₁₀ alkyl (preferablyC₁-C₆, more preferably C₁-C₃), optionally substituted C₆-C₁₄ aryl oroptionally substituted amino, or

NR′ where R′ is selected from

H,

O,

optionally substituted acyl group selected from H—C(O)—, C₁-C₁₀alkyl-C(O)— (preferably C₁-C₆ alkyl, more preferably C₁-C₃ alkyl), C₃-C₈cycloalkyl-C(O)—, C₆-C₁₄ aryl-C(O)—, heteroaryl-C(O)— where theheteroaryl group has from 2 to 10 carbon atoms and 1 to 4 heteroatomsselected from oxygen, nitrogen, selenium, and sulfur (including oxidesof sulfur, selenium and nitrogen) within the ring and heterocyclyl-C(O)—where the heterocyclyl group has from 1 to 8 carbon atoms and from 1 to4 heteroatoms selected from nitrogen, sulfur, oxygen, selenium orphosphorous within the ring). Examples of acyl groups include formylacetyl, propionyl, benzoyl (optionally substituted with methyl, methoxy,halogen, nitro, trifluoromethyl or cyano);

optionally substituted C₂-C₁₀ alkenyl group which may be straightchained or branched and have at least 1 or from 1-2 carbon to carbondouble bonds. Preferably optionally substituted C₂-C₆ alkenyl. Examplesof suitable optionally substituted alkenyl groups include ethenyl,n-propenyl, iso-propenyl, but-2-enyl, 1-propenyl, vinyl, nitrovinyl,cyano vinyl, or trifluorovinyl and styryl (optionally substituted withmethyl, methoxy, halogen, nitro, trifluoromethane or cyano);

optionally substituted C₁-C₁₀ alkyl, preferably C₁-C₆ alkyl, morepreferably C₁-C₃ alkyl. Examples of suitable alkyl groups includemethyl, ethyl, 1-hydroxyethyl, 1-thioethyl, methoxyiminomethyl,ethoxyiminomethyl, 1-(hydroxyimino)ethyl, 1-(hydroxyimino)propyl,1-hydrazinoethyl, 1-hydrazinopropyl, hydroxyiminomethyl, 2-oxopropyl,2-oxobutyl, 3-oxobutyl, 3-oxopentyl, nitromethyl, 1-nitromethyl, and2-nitroethyl;

optionally substituted C₆-C₁₄ aryl;

optionally substituted C₄-C₈ cycloalkenyl;

optionally substituted C₃-C₈ cycloalkyl;

optionally substituted heteroaryl having from 2 to 10 carbon atoms and 1to 4 heteroatoms selected from oxygen, nitrogen, selenium, and sulfur(including oxides of sulfur, selenium and nitrogen) within the ring

optionally substituted heterocyclyl having from 1 to 8 carbon atoms andfrom 1 to 4 heteroatoms selected from nitrogen, sulfur, oxygen, seleniumor phosphorous within the ring; or

optionally substituted sulfonyl selected from H—S(O)₂—, C₁-C₁₀alkyl-S(O)₂— (preferably C₁-C₆ alkyl, more preferably C₁-C₃ alkyl),C₃-C₈ cycloalkyl-S(O)₂—, C₆-C₁₄ aryl-S(O)₂—, heteroaryl-S(O)₂— where theheteroaryl group has from 2 to 10 carbon atoms and 1 to 4 heteroatomsselected from oxygen, nitrogen, selenium, and sulfur (including oxidesof sulfur, selenium and nitrogen) within the ring, andheterocyclyl-S(O)₂— where the heterocyclyl group has from 1 to 8 carbonatoms and from 1 to 4 heteroatoms selected from nitrogen, sulfur,oxygen, selenium or phosphorous within the ring. Examples of sulfonylgroups include methylsulfonyl, ethylsulfonyl, benzenesulfonyl(optionally substituted with methyl, methoxy, halogen, nitro,trifluoromethane or cyano), methoxycarbo, trifluoromethane;

In some embodiments Q is selected from the following groups:

H;

CN;

halogen, preferably Br or Cl;

trialkylsilyl, in which each alkyl group is independently C₁-C₁₀ alkyl(preferably C₁-C₆ alkyl, more preferably C₁-C₃ alkyl);

optionally substituted C₁-C₁₀ alkyl (preferably C₁-C₆ alkyl, morepreferably C₁-C₃ alkyl). Examples of suitable alkyl groups includemethyl, ethyl, propyl, butyl, aminoalkyl, oxyacylaminoalkyl andoxysulphonylaminoalkyl;

optionally substituted C₂-C₁₀ alkenyl group which may be straightchained or branched and have at least 1 or from 1-2 carbon to carbondouble bonds. Preferably optionally substituted C₂-C₆ alkenyl. Examplesof suitable optionally substituted alkenyl groups include ethenyl,n-propenyl, iso-propenyl, but-2-enyl, 1-propenyl, vinyl, nitrovinyl,cyano vinyl, or trifluorovinyl and styryl (optionally substituted withmethyl, methoxy, halogen, nitro, trifluoromethane or cyano);

optionally substituted C₂-C₁₀ alkynyl group having at least 1 or from1-2 carbon to carbon triple bonds. Preferably C₂-C₆ alkynyl. Examples ofsuitable alkynyl groups include 1-propynyl, ethynyl, propargyl,pent-2-ynyl, trimethylsilylethynyl and 2-alkylethynyl.

optionally substituted oxyacyl selected from HOC(O)—, C₁-C₁₀alkyl-OC(O)— (preferably preferably C₁-C₆ alkyl, more preferably C₁-C₃alkyl), C₃-C₈ cycloalkyl-OC(O)—, C₆-C₁₄ aryl-OC(O)—, heteroaryl-OC(O)—where the heteroaryl group has from 2 to 10 carbon atoms and 1 to 4heteroatoms selected from oxygen, nitrogen, selenium, and sulfur(including oxides of sulfur, selenium and nitrogen) within the ring, andheterocyclyl-OC(O)— where the heterocyclyl group has from 1 to 8 carbonatoms and from 1 to 4 heteroatoms selected from nitrogen, sulfur,oxygen, selenium or phosphorous within the ring. Examples of oxyacylgroups include methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl,butyloxycarbonyl, isobutyloxycarbonyl;

optionally substituted acyl group selected from H—C(O)—, C₁-C₁₀alkyl-C(O)— (preferably C₁-C₆ alkyl, more preferably C₁-C₃ alkyl), C₃-C₈cycloalkyl-C(O)—, C₆-C₁₄ aryl-C(O)—, heteroaryl-C(O)— where theheteroaryl group has from 2 to 10 carbon atoms and 1 to 4 heteroatomsselected from oxygen, nitrogen, selenium, and sulfur (including oxidesof sulfur, selenium and nitrogen) within the ring and heterocyclyl-C(O)—where the heterocyclyl group has from 1 to 8 carbon atoms and from 1 to4 heteroatoms selected from nitrogen, sulfur, oxygen, selenium orphosphorous within the ring). Examples of acyl groups include formylacetyl, propionyl, benzoyl (optionally substituted with methyl, methoxy,halogen, nitro, trifluoromethyl or cyano);

optionally substituted acylamino of the formula —NR*C(O)R* where each R*is independently hydrogen, C₁-C₁₀ alkyl (preferably C₁-C₆ alkyl, morepreferably C₁-C₃ alkyl), C₃-C₈ cycloalkyl, C₆-C₁₄ aryl, heteroarylhaving from 2 to 10 carbon atoms and 1 to 4 heteroatoms selected fromoxygen, nitrogen, selenium, and sulfur (including oxides of sulfur,selenium and nitrogen) within the ring and heterocyclyl having from 1 to8 carbon atoms and from 1 to 4 heteroatoms selected from nitrogen,sulfur, oxygen, selenium or phosphorous within the ring;

optionally substituted aminoacylamino, of the formula —NR*C(O)NR*R*where each R* is independently hydrogen, C₁-C₁₀ alkyl (preferably C₁-C₆alkyl, more preferably C₁-C₃ alkyl), C₃-C₈ cycloalkyl, C₆-C₁₄ aryl,heteroaryl having from 2 to 10 carbon atoms and 1 to 4 heteroatomsselected from oxygen, nitrogen, selenium, and sulfur (including oxidesof sulfur, selenium and nitrogen) within the ring and heterocyclylhaving from 1 to 8 carbon atoms and from 1 to 4 heteroatoms selectedfrom nitrogen, sulfur, oxygen, selenium or phosphorous within the ring;

OR″, where R″ is selected from H or an optionally substituted C₁-C₁₀alkyl (preferably C₁-C₆ alkyl, more preferably C₁-C₃ alkyl). Examples ofsuitable OR groups include hydroxy, methoxy, ethoxy, n-propoxy,iso-propoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy and1,2-dimethylbutoxy;

NR′R′, preferably R is selected from H, heteroaryl having from 2 to 10carbon atoms and 1 to 4 heteroatoms selected from oxygen, nitrogen,selenium, and sulfur (including oxides of sulfur, selenium and nitrogen)within the ring, amino, aminoC₁-C₁₀ alkyl (preferably C₁-C₆ alkyl, morepreferably C₁-C₃ alkyl), hydroxyl, hydroxyC₁-C₁₀ alkyl (preferably C₁-C₆alkyl, more preferably C₁-C₃ alkyl), C₁-C₁₀ alkoxy (preferably C₁-C₆alkoxy, more preferably C₁-C₃ alkoxy), C₁-C₁₀alkoxy C₁-C₁₀alkyl,oxyacyl, oxyacylalkyl, oxyacylamino, oxyacylaminoalkyl, guanidine,guanidinoalkyl or an optionally substituted C₁-C₁₀ alkyl group(preferably C₁-C₆ alkyl, more preferably C₁-C₃ alkyl). Examples ofsuitable N″R″ groups include NH₂, alkylamino, dialkylamino,heteroarylamino, aminoalkylamino, hydroxyalkylamino, alkoxyalkylamino,oxyacylalkylamino, oxyacylaminoalkylamino, guanidinoalkylamino;

SR″, preferably R″ is selected from H, heteroaryl having from 2 to 10carbon atoms and 1 to 4 heteroatoms selected from oxygen, nitrogen,selenium, and sulfur (including oxides of sulfur, selenium and nitrogen)within the ring, amino, aminoC₁-C₁₀ alkyl (preferably C₁-C₆ alkyl, morepreferably C₁-C₃ alkyl), hydroxyl, hydroxyC₁-C₁₀ alkyl (preferably C₁-C₆alkyl, more preferably C₁-C₃ alkyl), C₁-C₁₀ alkoxy (preferably C₁-C₆alkoxy, more preferably C₁-C₃ alkoxy), C₁-C₁₀alkoxy C₁-C₁₀alkyl,oxyacyl, oxyacylalkyl, oxyacylamino, oxyacylaminoalkyl, guanidine,guanidinoalkyl or an optionally substituted C₁-C₁₀ alkyl group(preferably C₁-C₆ alkyl, more preferably C₁-C₃ alkyl). Examples ofsuitable S′R″ groups include alkylthio, aminoalkylthio, heteroarylthio,aminoalkylthio, hydroxyalkylthio, alkoxyalkylthio, oxyacylalkylthio,oxyacylaminoalkylthio, guanidinoalkylthio;

hydrazine.

In the definitions of the groups X, R^(1A)-R^(1B), Q, L andR^(2A)-R^(2E), the term “optionally substituted” refers to a group whichmay or may not be further substituted or fused (so as to form acondensed polycyclic group) with one or more groups selected fromhydroxy, acyl, alkyl, alkoxy, alkenyl, alkenyloxy, alkynyl, alkynyloxy,amino, aminoacyl, thio, arylalkyl, arylalkoxy, aryl, aryloxy, acylamino,cyano, halogen, nitro, sulfo, phosphono, phosphorylamino, phosphinyl,heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, oxyacyl,oxime, oxime ether, hydrazone, —NHC(NH)NH₂, oxyacylamino,oxysulfonylamino, aminoacyloxy, trihalomethyl, trialkylsilyl,pentafluoroethyl, trifluoromethoxy, difluoromethoxy,trifluoromethanethio, trifluoroethenyl, mono- and di-alkylamino, mono-and di-(substituted alkyl)amino, mono- and di-arylamino, mono- anddi-heteroarylamino, mono- and di-heterocyclyl amino, and unsymmetricdi-substituted amines having different substituents selected from alkyl,aryl, heteroaryl and heterocyclyl, and the like.

In one embodiment R^(2D), R^(2C), and R^(2B) are methoxy and L is acarbonyl group (C═O).

Accordingly, in this embodiment the TPIs are represented by formula (Ia)or salts, solvates, or prodrugs thereof

wherein;

X represents O, S, SO, SO₂, Se, SeO, SeO₂ or NR where R is selected fromH, O, optionally substituted acyl, optionally substituted alkenyl,optionally substituted alkyl, optionally substituted aryl, optionallysubstituted cycloalkenyl, optionally substituted cycloalkyl, optionallysubstituted heteroaryl, optionally substituted heterocyclyl, andoptionally substituted sulfonyl;

R^(1A) and R^(1B) each independently represents H, carboxy, cyano,dihalomethoxy, halogen, hydroxy, nitro, pentahaloethyl, phosphorylamino,phosphono, phosphinyl, sulfo, trihaloethenyl, trihalomethanethio,trihalomethoxy, trihalomethyl, optionally substituted acyl, optionallysubstituted acylamino, optionally substituted acylimino, optionallysubstituted acyliminoxy, optionally substituted acyloxy, optionallysubstituted arylalkyl, optionally substituted arylalkoxy, optionallysubstituted alkenyl, optionally substituted alkenyloxy, optionallysubstituted alkoxy, optionally substituted alkyl, optionally substitutedalkynyl, optionally substituted alkynyloxy, optionally substitutedamino, optionally substituted aminoacyl, optionally substitutedaminoacyloxy, optionally substituted aminosulfonyl, optionallysubstituted aminothioacyl, optionally substituted aryl, optionallysubstituted aryloxy, optionally substituted cycloalkenyl, optionallysubstituted cycloalkyl, optionally substituted heteroaryl, optionallysubstituted heterocyclyl, optionally substituted oxyacyl, optionallysubstituted oxyacylamino, optionally substituted oxyacyloxy, optionallysubstituted oxyacylimino, optionally substituted oxysulfinylamino,optionally substituted oxysulfonylamino, optionally substitutedoxythioacyl, optionally substituted oxythioacyloxy, optionallysubstituted sulfinyl, optionally substituted sulfinylamino, optionallysubstituted sulfonyl, optionally substituted sulphonylamino, optionallysubstituted thio, optionally substituted thioacyl, optionallysubstituted thioacylamino, or R^(1A) and R^(1B) together form anoptionally substituted aryl, optionally substituted heterocyclyl,optionally substituted heteroaryl, optionally substituted cycloalkyl, oroptionally substituted cycloalkenyl;

R^(1C) represents C₁₋₃ alkoxy, C₁₋₃ alkylthio, C₁₋₃ alkylamino, or C₁₋₃dialkylamino;

R^(1D) represents hydroxy or amino;

R^(2A) and R^(2E) independently represents H, carboxy, cyano,dihalomethoxy, halogen, hydroxy, nitro, pentahaloethyl, phosphorylamino,phosphono, phosphinyl, sulfo, trihaloethenyl, trihalomethanethio,trihalomethoxy, trihalomethyl, optionally substituted acyl, optionallysubstituted acylamino, optionally substituted acylimino, optionallysubstituted acyliminoxy, optionally substituted acyloxy, optionallysubstituted arylalkyl, optionally substituted arylalkoxy, optionallysubstituted alkenyl, optionally substituted alkenyloxy, optionallysubstituted alkoxy, optionally substituted alkyl, optionally substitutedalkynyl, optionally substituted alkynyloxy, optionally substitutedamino, optionally substituted aminoacyl, optionally substitutedaminoacyloxy, optionally substituted aminosulfonyl, optionallysubstituted aminothioacyl, optionally substituted aryl, optionallysubstituted aryloxy, optionally substituted cycloalkenyl, optionallysubstituted cycloalkyl, optionally substituted heteroaryl, optionallysubstituted heterocyclyl, optionally substituted oxyacyl, optionallysubstituted oxyacylamino, optionally substituted oxyacyloxy, optionallysubstituted oxyacylimino, optionally substituted oxysulfinylamino,optionally substituted oxysulfonylamino, optionally substitutedoxythioacyl, optionally substituted oxythioacyloxy, optionallysubstituted sulfinyl, optionally substituted sulfinylamino, optionallysubstituted sulfonyl, optionally substituted sulphonylamino, optionallysubstituted thio, optionally substituted thioacyl, optionallysubstituted thioacylamino, or optionally substituted thioacyloxy; and

Q represents H, CN, halogen, trialkylsilyl, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted acyl, optionally substituted oxyacyl, optionallysubstituted acylamino, optionally substituted aminoacylamino, OR″, SR″or NR″R″, where each R″ independently represents, H, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted aryl, optionally substitutedheteroaryl, optionally substituted heterocyclyl, optionally substitutedacyl and optionally substituted oxyacyl, or NR″′NR″′, where each R″′independently represents H, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted aryl and optionally substituted heteroaryl.

In another embodiment, R^(1A), R^(1B), R^(2A) and R^(2E) represent H andR^(1C), R^(2B), R^(2C) and R^(2D) represents C₁₋₃ alkoxy.

Accordingly, in this embodiment the TPI is represented by formula (Ib)or salts, solvates or prodrugs thereof

wherein;

X represents O, S, SO, SO₂, Se, SeO, SeO₂ or NR where R is selected fromH, O, optionally substituted acyl, optionally substituted alkenyl,optionally substituted alkyl, optionally substituted aryl, optionallysubstituted cycloalkenyl, optionally substituted cycloalkyl, optionallysubstituted heteroaryl, optionally substituted heterocyclyl, andoptionally substituted sulfonyl;

R^(1C) represents C₁₋₃ alkoxy;

R^(1D) represents hydroxy or amino;

-   -   Q represents H, CN, halogen, trialkylsilyl, optionally        substituted alkyl, optionally substituted alkenyl, optionally        substituted alkynyl, optionally substituted acyl, optionally        substituted oxyacyl, optionally substituted acylamino,        optionally substituted aminoacylamino, OR″, SR″ or NR″R″, where        each R″ independently represents, H, optionally substituted        alkyl, optionally substituted alkenyl, optionally substituted        alkynyl, optionally substituted aryl, optionally substituted        heteroaryl, optionally substituted acyl and optionally        substituted oxyacyl, or NR″′NR″′, where each R″′ independently        represents H, optionally substituted alkyl, optionally        substituted alkenyl, optionally substituted alkynyl, optionally        substituted aryl and optionally substituted heteroaryl.

In a preferred embodiment R^(1C) represents methoxy.

For the compounds represented by formulae I, Ia and Ib, X is preferablyselected from O, S and NR. More preferably X is O or NR and mostpreferably X is O.

Accordingly, in another embodiment the TPI is represented by formula II:

wherein;

R^(1A) and R^(1B) each independently represents H, carboxy, cyano,dihalomethoxy, halogen, hydroxy, nitro, pentahaloethyl, phosphorylamino,phosphono, phosphinyl, sulfo, trihaloethenyl, trihalomethanethio,trihalomethoxy, trihalomethyl, optionally substituted acyl, optionallysubstituted acylamino, optionally substituted acylimino, optionallysubstituted acyliminoxy, optionally substituted acyloxy, optionallysubstituted arylalkyl, optionally substituted arylalkoxy, optionallysubstituted alkenyl, optionally substituted alkenyloxy, optionallysubstituted alkoxy, optionally substituted alkyl, optionally substitutedalkynyl, optionally substituted alkynyloxy, optionally substitutedamino, optionally substituted aminoacyl, optionally substitutedaminoacyloxy, optionally substituted aminosulfonyl, optionallysubstituted aminothioacyl, optionally substituted aryl, optionallysubstituted aryloxy, optionally substituted cycloalkenyl, optionallysubstituted cycloalkyl, optionally substituted heteroaryl, optionallysubstituted heterocyclyl, optionally substituted oxyacyl, optionallysubstituted oxyacylamino, optionally substituted oxyacyloxy, optionallysubstituted oxyacylimino, optionally substituted oxysulfinylamino,optionally substituted oxysulfonylamino, optionally substitutedoxythioacyl, optionally substituted oxythioacyloxy, optionallysubstituted sulfinyl, optionally substituted sulfinylamino, optionallysubstituted sulfonyl, optionally substituted sulphonylamino, optionallysubstituted thio, optionally substituted thioacyl, optionallysubstituted thioacylamino, or R^(1A) and R^(1B) together form anoptionally substituted aryl, optionally substituted heterocyclyl,optionally substituted heteroaryl, optionally substituted cycloalkyl, oroptionally substituted cycloalkenyl;

R^(1C) represents C₁₋₃ alkoxy, C₁₋₃ alkylthio, C₁₋₃ alkylamino, or C₁₋₃dialkylamino;

R^(1D) represents hydroxy or amino;

L represents C═O, O, S, SO, SO₂, Se, SeO, SeO₂, C═NZ′, or NR′ where Z′is H, optionally substituted alkyl, optionally substituted aryl oroptionally substituted amino; and where R′ is selected from H, O,optionally substituted acyl, optionally substituted alkenyl, optionallysubstituted alkyl, optionally substituted aryl, optionally substitutedcycloalkenyl, optionally substituted cycloalkyl, optionally substitutedheteroaryl, optionally substituted heterocyclyl, or optionallysubstituted sulfonyl;

R^(2A)-R^(2E) each independently represents H, carboxy, cyano,dihalomethoxy, halogen, hydroxy, nitro, pentahaloethyl, phosphorylamino,phosphono, phosphinyl, sulfo, trihaloethenyl, trihalomethanethio,trihalomethoxy, trihalomethyl, optionally substituted acyl, optionallysubstituted acylamino, optionally substituted acylimino, optionallysubstituted acyliminoxy, optionally substituted acyloxy, optionallysubstituted arylalkyl, optionally substituted arylalkoxy, optionallysubstituted alkenyl, optionally substituted alkenyloxy, optionallysubstituted alkoxy, optionally substituted alkyl, optionally substitutedalkynyl, optionally substituted alkynyloxy, optionally substitutedamino, optionally substituted aminoacyl, optionally substitutedaminoacyloxy, optionally substituted aminosulfonyl, optionallysubstituted aminothioacyl, optionally substituted aryl, optionallysubstituted aryloxy, optionally substituted cycloalkenyl, optionallysubstituted cycloalkyl, optionally substituted heteroaryl, optionallysubstituted heterocyclyl, optionally substituted oxyacyl, optionallysubstituted oxyacylamino, optionally substituted oxyacylimino,optionally substituted oxyacyloxy, optionally substitutedoxysulfinylamino, optionally substituted oxysulfonylamino, optionallysubstituted oxythioacyl, optionally substituted oxythioacyloxy,optionally substituted sulfinyl, optionally substituted sulfinylamino,optionally substituted sulfonyl, optionally substituted sulphonylamino,optionally substituted thio, optionally substituted thioacyl, optionallysubstituted thioacylamino, or optionally substituted thioacyloxy; or anyof R^(2A) and R^(2B), R^(2B) and R^(2C), R^(2C) and R^(2D), and R^(2D)and R^(2E), together form an optionally substituted aryl, optionallysubstituted heterocyclyl, optionally substituted heteroaryl, optionallysubstituted cycloalkyl, or optionally substituted cycloalkenyl; and

Q represents H, CN, halogen, trialkylsilyl, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted acyl, optionally substituted oxyacyl, optionallysubstituted acylamino, optionally substituted aminoacylamino, OR″, SR″or NR″R″, where each R″ independently represents, H, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted heterocyclyl, optionallysubstituted aryl, optionally substituted heteroaryl, optionallysubstituted acyl and optionally substituted oxyacyl, or NR″′NR″′, whereeach R″′ independently represents H, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl and optionally substituted heteroaryl.

In this embodiment it is preferred that L is a carbonyl group (C═O).Also, preferably at least one of R^(2D), R^(2C) or R^(2B) represents ahydroxy or C₁₋₃ alkoxy group. More preferably when X═O, L is a carbonylgroup an R^(2D), R^(2C) and R^(2B) represent methoxy. Even morepreferably when X═O, L is a carbonyl group, R^(2D), R^(2C), and R^(2B)represent methoxy and R^(1A), R^(1B), R^(2A), R^(2E) are H.

Furthermore, for the compounds of formula (I), (Ia), (Ib) and (II) it ispreferred that Q represents H, CN, optionally substituted C₂₋₄ alkynyl,optionally substituted C₂₋₆ alkenyl, optionally substituted C₁₋₄ alkyl,hydroxy, optionally substituted oxyacyl, NR″R″, SR″ (where each R″ isindependently H, optionally substituted C₁₋₄alkyl, optionallysubstituted heterocyclyl, optionally substituted heteroaryl), NR″′NR″′(where each R″′ is independently H, C₁₋₃ alkyl), optionally substitutedacylamino, or halogen.

In some embodiments Q is independently selected from the followinggroups:

H;

CN;

halogen, preferably Br or Cl;

alkyl group, preferably methyl, ethyl, propyl, butyl;

substituted alkyl group, preferably amino, oxyacylaminoalkyl andoxysulphonylaminoalkyl;

optionally substituted alkenyl, preferably ethenyl, 2-alkylethenyl,2-oxyacylethenyl, 2-aminoacylethenyl;

optionally substituted alkynyl, preferably ethynyl, 2-alkylethynyl;

optionally substituted oxyacyl;

OR″, preferably hydroxy, methoxy, ethoxy;

NR″R″, preferably NH₂, alkylamino, dialkylamino, heteroarylamino,aminoalkylamino, hydroxyalkylamino, alkoxyalkylamino, oxyacylalkylamino,oxyacylaminoalkylamino, guanidinoalkylamino;

SR″, preferably alkylthio, aminoalkylthio, heteroarylthio,aminoalkylthio, hydroxyalkylthio, alkoxyalkylthio, oxyacylalkylthio,oxyacylaminoalkylthio, guanidinoalkylthio; hydrazine.

In a further preferred embodiment the TPI for use in the present methodis a compound of formula (III) or a salt, solvate or prodrug thereof

In an embodiment, the compound of formula (I), (Ia), (Ib), (II) or (III)is a prodrug selected from an ester, an acetate, a phosphate ester or anamide prodrug. In another embodiment, the compound of formula (I) (Ia),(Ib), (II) or (III) is a phosphate prodrug. In a particular embodiment,R^(1D) is hydroxy and the prodrug is a phosphate ester of the hydroxygroup. Preferably, the phosphate ester is a disodium phosphate ester.

The compound of formula (III)(2-Methyl-7-hydroxy-3-(3,4,5-trimethoxybenzoyl)-6-methoxybenzofuran) canbe prepared by the synthetic methodology described in PCT/AU2007/000101(WO 07/087684).

The compounds of formula I, Ia, Ib, II or III have been observed to bepotent tubulin polymerisation inhibitors (TPIs). An important aspect ofthe compounds of formulae I, Ia, Ib, II and III is the combination ofthe specific C-6 and C-7 substituents together with the C-2 Q-group(especially C-2 methyl) which appears to confer greater potency andselectivity when compared to other structurally related TPI compounds.In these compounds selectivity is not simply reliant on thepredisposition of tumour vasculature towards collapse when challengedwith the VDA but on a capacity of the VDA to distinguish between tumourendothelial cells and normal endothelial cells. Normal endothelialcells, found in healthy tissues, are in a “quiescent” state and tumourendothelial cells are in an “activated” state. Most VDAs do notdistinguish between these two states, for example, Combretastatin A4(CA4) is equally potent against quiescent and activated endothelialcells. However, the compounds of formulae I, Ia, Ib, II and particularlyIII show selectivity towards tumor endothelial cells (activated) overnormal endothelial cells (quiescent).

In some embodiments, the TPI for use in the present method is a compoundof formula I, Ia, Ib or II or a salt, solvate or prodrug thereof whereinR^(1C) is C₁₋₃ alkoxy, R^(1D) is hydroxyl and Q is optionallysubstituted C₁₋₁₀ (or C₁₋₆ or C₁-3) alkyl.

The TPI compounds of formula I, Ia, Ib, II or III may be prepared byknown methods including those disclosed in WO 02/060872 and WO 07/087684which are incorporated herein by reference.

It will be appreciated that the TPIs and compounds of formula I, Ia, Ib,II, or III can be administered to a subject as a pharmaceuticallyacceptable salt thereof. Suitable pharmaceutically acceptable saltsinclude, but are not limited to salts of pharmaceutically acceptableinorganic acids such as hydrochloric, sulphuric, phosphoric, nitric,carbonic, boric, sulfamic, and hydrobromic acids, or salts ofpharmaceutically acceptable organic acids such as acetic, propionic,butyric, tartaric, maleic, hydroxymaleic, fumaric, maleic, citric,lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic,methanesulphonic, toluenesulphonic, benezenesulphonic, salicyclicsulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic,lauric, pantothenic, tannic, ascorbic and valeric acids.

Base salts include, but are not limited to, those formed withpharmaceutically acceptable cations, such as sodium, potassium, lithium,calcium, magnesium, ammonium and alkylammonium. In particular, thepresent invention includes within its scope cationic salts eg sodium orpotassium salts, or alkyl esters (eg methyl, ethyl) of the phosphategroup.

It will also be appreciated that any compound that is a prodrug of a TPIor a compound of formula I, Ia, Ib, II, and III are also within thescope of the invention. The term “pro-drug” is used in its broadestsense and encompasses those derivatives that are converted in vivo tothe compound (for instance, a compound of formulae I, Ia, Ib, II, andIII). Such derivatives would readily occur to those skilled in the art,and include, for example, compounds where the free hydroxy group (forinstance at C-7 position or RID) is converted into an ester, such as anacetate or phosphate ester, or where a free amino group (for instance atC-7 position or R^(1D)) is converted into an amide (e.g., α-aminoacidamide). Procedures for esterifying, for example, acylating, thecompounds are well known in the art and may include treatment of thecompound with an appropriate carboxylic acid, anhydride or chloride inthe presence of a suitable catalyst or base. A particularly preferredprodrug is a disodium phosphate ester. The disodium phosphate ester (inparticular a C-7 disodium phosphate ester of a compound of formula III)of the compound may be useful in increasing the solubility of thecompounds. This, for instance, may allow for delivery of the compound ina benign vehicle like saline. The disodium phosphate ester may beprepared in accordance with the methodology described in Pettit, G. R.,et al, Anticancer Drug Des., 1995, 10, 299. Other texts which generallydescribe prodrugs (and the preparation thereof) include: Design ofProdrugs, 1985, H. Bundgaard (Elsevier); The Practice of MedicinalChemistry, 1996, Camille G. Wermuth et al., Chapter 31 (Academic Press);and A Textbook of Drug Design and Development, 1991, Bundgaard et al.,Chapter 5, (Harwood Academic Publishers).

In some embodiments, the TPI for use in the present method is a compoundof formula (IV)

wherein, X, R^(1A)-R^(1C) and R^(2A)-R^(2E), L and Q are as defined informula I, Ia, Ib, II or III, and R^(1D) is OR³ or NHR³, and R³ is H oran ester. When R³ is an ester, the ester may consist of a carbonyladjacent to an ether linkage (such as an acetate ester), or may be aninorganic ester (such as a phosphate, sulfate, nitrate or borate ester).In some embodiments, the ester is an acetate or a phosphate ester. Aparticularly preferred ester is a disodium phosphate ester.

The compounds of formulae I, Ia, Ib, II, III and IV (or a salt orprodrug thereof) may be in crystalline form either as the free compoundor as a solvate (e.g. hydrate) and it is intended that both forms arewithin the scope of the present invention. Methods of solvation aregenerally known within the art.

Chemical Definitions

“Alkyl” refers to monovalent alkyl groups which may be straight chainedor branched and preferably have from 1 to 10 carbon atoms or morepreferably 1 to 6 carbon atoms, and even more preferably 1 to 3 carbonatoms. Examples of such alkyl groups include methyl, ethyl, n-propyl,iso-propyl, n-butyl, iso-butyl, n-hexyl, and the like.

“Alkylene” refers to divalent alkyl groups preferably having from 1 to10 carbon atoms and more preferably 1 to 6 carbon atoms, and even morepreferably 1 to 3 carbon atoms. Examples of such alkylene groups includemethylene (—CH₂—), ethylene (—CH₂CH₂—), and the propylene isomers (e.g.,—CH₂CH₂CH₂— and —CH(CH₃)CH₂—), and the like.

“Aryl” refers to an unsaturated aromatic carbocyclic group having asingle ring (eg., phenyl) or multiple condensed rings (eg., naphthyl oranthryl), preferably having from 6 to 14 carbon atoms. Examples of arylgroups include phenyl, naphthyl and the like.

“Arylene” refers to a divalent aryl group wherein the aryl group is asdescribed above.

“Aryloxy” refers to the group aryl-O— wherein the aryl group is asdescribed above.

“Arylalkyl” refers to -alkylene-aryl groups preferably having from 1 to10 carbon atoms in the alkylene moiety and from 6 to 10 carbon atoms inthe aryl moiety. Such arylalkyl groups are exemplified by benzyl,phenethyl and the like.

“Arylalkoxy” refers to the group arylalkyl-O— wherein the arylalkylgroup are as described above. Such arylalkoxy groups are exemplified bybenzyloxy and the like.

“Alkoxy” refers to the group alkyl-O— where the alkyl group is asdescribed above. Examples include, methoxy, ethoxy, n-propoxy,iso-propoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy,1,2-dimethylbutoxy, and the like.

“Alkenyl” refers to a monovalent alkenyl group which may be straightchained or branched and preferably have from 2 to 10 carbon atoms andmore preferably 2 to 6 carbon atoms and have at least 1 and preferablyfrom 1-2, carbon to carbon, double bonds. Examples include ethenyl(—CH═CH₂), n-propenyl (—CH₂CH═CH₂), iso-propenyl (—C(CH₃)═CH₂),but-2-enyl (—CH₂CH═CHCH₃), and the like.

“Alkenyloxy” refers to the group alkenyl-O— wherein the alkenyl group isas described above.

“Alkenylene” refers to divalent alkenyl groups preferably having from 2to 8 carbon atoms and more preferably 2 to 6 carbon atoms. Examplesinclude ethenylene (—CH═CH—), and the propenylene isomers (e.g.,—CH₂CH═CH— and —C(CH₃)═CH—), and the like.

“Alkynyl” refers to alkynyl groups preferably having from 2 to 10 carbonatoms and more preferably 2 to 6 carbon atoms and having at least 1, andpreferably from 1-2, carbon to carbon, triple bonds. Examples of alkynylgroups include ethynyl (—C≡CH), propargyl (—CH₂C≡CH), pent-2-ynyl(—CH₂C≡CCH₂—CH₃), and the like.

“Alkynyloxy” refers to the group alkynyl-O— wherein the alkynyl groupsis as described above.

“Alkynylene” refers to the divalent alkynyl groups preferably havingfrom 2 to 8 carbon atoms and more preferably 2 to 6 carbon atoms.Examples include ethynylene (—C≡C—), propynylene (—CH₂—C≡C—), and thelike.

“Acyl” refers to groups H—C(O)—, alkyl-C(O)—, cycloalkyl-C(O)—,aryl-C(O)—, heteroaryl-C(O)— and heterocyclyl-C(O)—, where alkyl,cycloalkyl, aryl, heteroaryl and heterocyclyl are as described herein.

“Oxyacyl” refers to groups HOC(O)—, alkyl-OC(O)—, cycloalkyl-OC(O)—,aryl-OC(O)—, heteroaryl-OC(O)—, and heterocyclyl-OC(O)—, where alkyl,cycloalkyl, aryl, heteroaryl and heterocyclyl are as described herein.

“Amino” refers to the group —NR*R* where each R* is independentlyhydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl andwhere each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is asdescribed herein.

“Aminoacyl” refers to the group —C(O)NR*R* where each R* isindependently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, andheterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl andheterocyclyl is as described herein.

“Aminoacylamino” refers to the group —NR*C(O)NR*R* where each R* isindependently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, andheterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl andheterocyclyl is as described herein.

“Acylamino” refers to the group —NR*C(O)R* where each R* isindependently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl andheterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl, andheterocyclyl are as described herein.

“Acyloxy” refers to the groups —OC(O)-alkyl, —OC(O)-aryl,—C(O)O-heteroaryl, and —C(O)O-heterocyclyl where alkyl, aryl, heteroaryland heterocyclyl are as described herein.

“Aminoacyloxy” refers to the groups —OC(O)NR*-alkyl, —OC(O)NR*-aryl,—OC(O)NR*-heteroaryl, and —OC(O)NR*-heterocyclyl where R* isindependently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, andheterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl andheterocyclyl is as described herein.

“Oxyacylamino” refers to the groups —NR*C(O)O-alkyl, —NR*C(O)O-aryl,—NR*C(O)O-heteroaryl, and NR*C(O)O-heterocyclyl where R* isindependently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, andheterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl andheterocyclyl is as described herein.

“Oxyacyloxy” refers to the groups —OC(O)O-alkyl, —O—C(O)O-aryl, —OC(O)O—heteroaryl, and —OC(O)O-heterocyclyl where alkyl, cycloalkyl, aryl,heteroaryl, and heterocyclyl are as described herein.

“Acylimino” refers to the groups —C(NR*)—R* where each R* isindependently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl andheterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl, andheterocyclyl are as described herein.

“Acyliminoxy” refers to the groups —O—C(NR*)—R* where each R* isindependently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl andheterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl, andheterocyclyl are as described herein.

“Oxyacylimino” refers to the groups —C(NR*)—OR* where each R* isindependently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl andheterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl, andheterocyclyl are as described herein.

“Cycloalkyl” refers to cyclic alkyl groups having a single cyclic ringor multiple condensed rings, preferably incorporating 3 to 8 carbonatoms. Such cycloalkyl groups include, by way of example, single ringstructures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cyclooctyl, and the like, or multiple ring structures such asadamantanyl, and the like.

“Cycloalkenyl” refers to cyclic alkenyl groups having a single cyclicring and at least one point of internal unsaturation, preferablyincorporating 4 to 8 carbon atoms. Examples of suitable cycloalkenylgroups include, for instance, cyclobut-2-enyl, cyclopent-3-enyl,cyclohex-4-enyl, cyclooct-3-enyl and the like.

“Halo” or “halogen” refers to fluoro, chloro, bromo and iodo.

“Heteroaryl” refers to a monovalent aromatic heterocyclic group whichfulfils the Hückel criteria for aromaticity (ie. contains 4n+2πelectrons) and preferably has from 2 to 10 carbon atoms and 1 to 4heteroatoms selected from oxygen, nitrogen, selenium, and sulfur withinthe ring (and includes oxides of sulfur, selenium and nitrogen). Suchheteroaryl groups can have a single ring (eg., pyridyl, pyrrolyl orN-oxides thereof or furyl) or multiple condensed rings (eg.,indolizinyl, benzoimidazolyl, coumarinyl, quinolinyl, isoquinolinyl orbenzothienyl).

“Heterocyclyl” refers to a monovalent saturated or unsaturated grouphaving a single ring or multiple condensed rings, preferably from 1 to 8carbon atoms and from 1 to 4 heteroatoms selected from nitrogen, sulfur,oxygen, selenium or phosphorous within the ring. The most preferredheteroatom is nitrogen.

Examples of heterocyclyl and heteroaryl groups include, but are notlimited to, oxazole, pyrrole, imidazole, pyrazole, pyridine, pyrazine,pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine,quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine,quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline,phenanthridine, acridine, phenanthroline, isothiazole, phenazine,isoxazole, isothiazole, phenoxazine, phenothiazine, imidazolidine,imidazoline, piperidine, piperazine, indoline, phthalimide,1,2,3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene,thiazole, thiadiazoles, oxadiazole, oxatriazole, tetrazole,thiazolidine, thiophene, benzo[b]thiophene, morpholino, piperidinyl,pyrrolidine, tetrahydrofuranyl, triazole, and the like.

“Heteroarylene” refers to a divalent heteroaryl group wherein theheteroaryl group is as described above.

“Heterocyclylene” refers to a divalent heterocyclyl group wherein theheterocyclyl group is as described above.

“Thio” refers to groups H—S—, alkyl-S—, cycloalkyl-S—, aryl-S—,heteroaryl-S—, and heterocyclyl-S—, where alkyl, cycloalkyl, aryl,heteroaryl and heterocyclyl are as described herein.

“Thioacyl” refers to groups H—C(S)—, alkyl-C(S)—, cycloalkyl-C(S)—,aryl-C(S)—, heteroaryl-C(S)—, and heterocyclyl-C(S)—, where alkyl,cycloalkyl, aryl, heteroaryl and heterocyclyl are as described herein.

“Oxythioacyl” refers to groups HO—C(S)—, alkylO—C(S)—,cycloalkylO—C(S)—, arylO—C(S)—, heteroarylO—C(S)—, andheterocyclylO—C(S)—, where alkyl, cycloalkyl, aryl, heteroaryl andheterocyclyl are as described herein.

“Oxythioacyloxy” refers to groups HO—C(S)—O—, alkylO—C(S)—O—,cycloalkylO—C(S)—O—, arylO—C(S)—O—, heteroarylO—C(S)—O—, andheterocyclylO—C(S)—O—, where alkyl, cycloalkyl, aryl, heteroaryl andheterocyclyl are as described herein.

“Phosphorylamino” refers to the groups —NR*—P(O)(R**)(OR***) where R*represents H, alkyl, cycloalkyl, alkenyl, or aryl, R** represents OR***or is hydroxy or amino and R*** is alkyl, cycloalkyl, aryl or arylalkyl,where alkyl, amino, alkenyl, aryl, cycloalkyl, and arylalkyl are asdescribed herein.

“Thioacyloxy” refers to groups H—C(S)—O—, alkyl-C(S)—O—,cycloalkyl-C(S)—O—, aryl-C(S)—O—, heteroaryl-C(S)—O—, andheterocyclyl-C(S)—O—, where alkyl, cycloalkyl, aryl, heteroaryl, andheterocyclyl are as described herein.

“Sulfinyl” refers to groups H—S(O)—, alkyl-S(O)—, cycloalkyl-S(O)—,aryl-S(O)—, heteroaryl-S(O)—, and heterocyclyl-S(O)—, where alkyl,cycloalkyl, aryl, heteroaryl and heterocyclyl are as described herein.

“Sulfonyl” refers to groups H—S(O)₂—, alkyl-S(O)₂—, cycloalkyl-S(O)₂—,aryl-S(O)₂—, heteroaryl-S(O)₂—, and heterocyclyl-S(O)₂—, where alkyl,cycloalkyl, aryl, heteroaryl and heterocyclyl are as described herein.

“Sulfinylamino” refers to groups H—S(O)—NR*—, alkyl-S(O)—NR*—,cycloalkyl-S(O)—NR*—, aryl-S(O)—NR*—, heteroaryl-S(O)—NR*—, andheterocyclyl-S(O)—NR*—, where R* is independently hydrogen, alkyl,cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl,cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.

“Sulfonylamino” refers to groups H—S(O)₂—NR*—, alkyl-S(O)₂—NR*—,cycloalkyl-S(O)₂—NR*—, aryl-S(O)₂—NR*—, heteroaryl-S(O)₂—NR*—, andheterocyclyl-S(O)₂—NR*—, where R* is independently hydrogen, alkyl,cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl,cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.

“Oxysulfinylamino” refers to groups HO—S(O)—NR*—, alkylO—S(O)—NR*—,cycloalkylO—S(O)—NR*—, arylO—S(O)—NR*—, heteroarylO—S(O)—NR*—, andheterocyclylO—S(O)—NR*—, where R* is independently hydrogen, alkyl,cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl,cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.

“Oxysulfonylamino” refers to groups HO—S(O)₂—NR*—, alkylO—S(O)₂—NR*—,cycloalkylO—S(O)₂—NR*—, arylO—S(O)₂—NR*—, heteroarylO—S(O)₂—NR*—, andheterocyclylO—S(O)₂—NR*—, where R* is independently hydrogen, alkyl,cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl,cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.

“Aminothioacyl” refers to groups R*R*N—C(S)—, where each R* isindependently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, andheterocyclic and where each of alkyl, cycloalkyl, aryl, heteroaryl andheterocyclyl is as described herein.

“Thioacylamino” refers to groups H—C(S)—NR*—, alkyl-C(S)—NR*—,cycloalkyl-C(S)—NR*—, aryl-C(S)—NR*—, heteroaryl-C(S)—NR*—, andheterocyclyl-C(S)—NR*—, where R* is independently hydrogen, alkyl,cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl,cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.

“Aminosulfinyl” refers to groups R*R*N—S(O)—, where each R* isindependently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, andheterocyclic and where each of alkyl, cycloalkyl, aryl, heteroaryl andheterocyclyl is as described herein.

“Aminosulfonyl” refers to groups R*R*N—S(O)₂—, where each R* isindependently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, andheterocyclic and where each of alkyl, cycloalkyl, aryl, heteroaryl andheterocyclyl is as described herein.

In this specification “optionally substituted” is taken to mean that agroup may or may not be further substituted or fused (so as to form acondensed polycyclic group) with one or more groups selected fromhydroxy, acyl, alkyl, alkoxy, alkenyl, alkenyloxy, alkynyl, alkynyloxy,amino, aminoacyl, thio, arylalkyl, arylalkoxy, aryl, aryloxy, acylamino,cyano, halogen, nitro, sulfo, phosphono, phosphorylamino, phosphinyl,heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, oxyacyl,oxime, oxime ether, hydrazone, —NHC(NH)NH₂, oxyacylamino,oxysulfonylamino, aminoacyloxy, trihalomethyl, trialkylsilyl,pentafluoroethyl, trifluoromethoxy, difluoromethoxy,trifluoromethanethio, trifluoroethenyl, mono- and di-alkylamino, mono-and di-(substituted alkyl)amino, mono- and di-arylamino, mono- anddi-heteroarylamino, mono- and di-heterocyclyl amino, and unsymmetricdi-substituted amines having different substituents selected from alkyl,aryl, heteroaryl and heterocyclyl, and the like. An optionallysubstituted amino group may also include amino acid and peptideresidues.

mTOR Inhibitors

In one embodiment of the invention, the method of the invention ispredictive of patient response to treatment with a vascular disruptingagent in combination with an mTOR inhibitor. The mechanistic target ofrapamycin (mTOR serine/threonine kinase), also known as mammalian targetof rapamycin, mTOR, or FK506-binding protein 12-rapamycin-associatedprotein 1 (FRAP1), is a serine/threonine protein kinase that regulatescell growth, cell proliferation, cell motility, cell survival, proteinsynthesis, and transcription. MTOR belongs to the phosphatidylinositol3-kinase-related kinase protein family. PI3K/Akt-dependentphosphorylation signals through tuberin, the protein product of theTSC1/TSC2 complex, leading to mTOR activation. mTOR subsequentlyphosphorylates downstream targets, causing initiation of proteintranslation. Accordingly, any agent that inhibits the activation ofmTOR, causing down-regulation of its downstream targets, is encompassedby the meaning of “mTOR inhibitor” as used herein.

Examples of mTOR inhibitors include BEZ235 (NVP-BEZ235), deforolimus (AP23573, MK-8669), PI-103, rapamycin (Sirolimus, Rapamune), temsirolimus(Toricel, CCI-779), everolimus (Afinitor, RAD001, Certican), ABT 578,SAR 543 and AP 23841. In one particular embodiment, the mTOR inhibitoris everolimus (Afinitor).

Dosing and Administration

In one embodiment, a patient identified as having an increasedlikelihood of responding to treatment is administered a vasculardisrupting agent. Daily dosages for the vascular disrupting agent will,of course, vary depending on a variety of factors, e.g., the compoundchosen, the particular condition to be treated and the desired effect.In general, however, satisfactory results are achieved on administrationat daily dosage rates of about 0.05 to 20 mg/kg per day, particularly 1to 20 mg/kg per day, e.g. 0.4 to 16 mg/kg per day, as a single dose orin divided doses. The vascular disrupting agent may be administered byany conventional route, in particular parenterally, e.g., in the form ofinjectable solutions or suspensions, or enterally, e.g., orally, e.g.,in the form of tablets, capsules, drink solutions. Suitable unit dosageforms for oral administration comprise from about 0.02 to 50 mg activeingredient, usually 0.1 to 30 mg and 2 to 25 mg, 4 to 20 mg e.g.together with one or more pharmaceutically acceptable diluents orcarriers therefore.

For instance, an administration regime may include adding the vasculardisrupting agent (e.g., compound of formula I, Ia, Ib, II, or III) at anassigned dose level by I.V. on days 1 and 8 (of a 21 day cycle). In thisembodiment the compound of formula (III) may be dosed at a level ofbetween 4 to 16 mg/kg.

Thus, while the skilled person will readily be able to determinesuitable doses of the vascular disrupting agent, in one embodiment,BNC105P is administered at a dosage of about 8 mg/m² to about 16 mg/m².In one particular embodiment, BNC105P is administered at a dosage of 16mg/m² on day 1 and day 8 of a 21 day treatment cycle. As understood inthe art, the patient may receive multiple cycles of treatment.

In one embodiment, the patient is treated with a combination therapycomprising a vascular disrupting agent and another therapeutic. Asunderstood in the art, the terms “combination therapy”, “combinationtreatment”, or “pharmaceutical combination” refer to the use of morethan one medication or other therapy (vs. monotherapy, which is anytherapy taken alone), to treat a single disease. A “Pharmaceuticalcombination” therapy, for example, may be achieved byprescribing/administering separate drugs, or, where available, dosageforms that contain more than one active ingredient (such as fixed-dosecombinations).

The methods and uses of the present invention encompass theadministration of the an additional therapeutic agent or a salt, solvateor prodrug thereof (combination partner a) in combination with avascular disrupting agent or a salt, solvate or prodrug thereof(combination partner b) to a single patient, and is intended to includetreatment regimens in which the agents are not necessarily administeredby the same route of administration or at the same time. Accordingly,combination partners (a) and (b) may be administered together, one afterthe other or separately in one combined unit dosage form or in twoseparate unit dosage forms. The unit dosage form may also be a fixedcombination such as a pharmaceutical composition which comprises bothpartner (a) (or a salt, solvate or prodrug thereof) and partner (b) (ora salt, solvate or prodrug thereof).

In particular, a therapeutically effective amount of each of thecombination partner of the combination of the invention may beadministered simultaneously or sequentially and in any order, and thecomponents may be administered separately or as a fixed combination.

For example, the method of the invention may comprise: (i)administration of partner (a) in free or pharmaceutically acceptablesalt form; and (ii) administration of partner (b) in free orpharmaceutically acceptable salt form, simultaneously or sequentially inany order, in jointly therapeutically effective amounts, preferably insynergistically effective amounts, e.g., in daily or intermittentdosages corresponding to the amounts described herein. The individualcombination partners of the combination of the invention may beadministered separately at different times during the course of therapyor concurrently in divided or single combination forms. Furthermore, theterm administering also encompasses the use of a pro-drug of acombination partner that converts in vivo to the combination partner assuch.

In one embodiment, the patient is treated with a combination of avascular disrupting agent and an mTOR inhibitor. Examples of mTORinhibitors suitable for use in the present invention include BEZ235(NVP-BEZ235), deforolimus (AP 23573, MK-8669), PI-103, rapamycin(Sirolimus, Rapamune), temsirolimus (Toricel, CCI-779), everolimus(Afinitor, RAD001, Certican), ABT 578, SAR 543 and AP 23841. In oneparticular embodiment, the mTOR inhibitor is everolimus (Afinitor). Inone particular embodiment, the patient is administered a vasculardisrupting agent in combination with everolimus.

In one embodiment, everolimus is administered daily to the patient.

In another embodiment, BNC105P is administered to the patient in weeklydoses. In one particular embodiment, everolimus is administered daily tothe patient, and BNC105P is administered to the patient after about 5,6, 7, 8 or 9 days of everolimus administration and again at about 13,14, 15, 16, or 17 days of everolimus administration. In one embodiment,the everolimus is administered daily at a dosage of 10 mg.

Additional Therapies

The methods of the present invention may utilise the combination of avascular disrupting agent in conjunction with other therapeutic agentsand treatment modalities such as tumor irradiation. For example, thecombination therapy of the present invention may be used in conjunctionwith another chemotherapeutic, antibody and or immunotherapeutic that issuitable for administration to a patient for the treatment of cancer.

Examples of therapeutic agents that may be administered in conjunctionwith the combination of a vascular disrupting agent include mTORinhibitors, tyrosine kinase inhibitors, such as VEGF-directed tyrosinekinase inhibitors and proteasome inhibitors. By way of example, tyrosinekinase inhibitors include sunitinib (Sutent), sorafenib (Nexavar),axitinib (Inlyta) and pazopanib (Votrient). Another therapeutic agentused in the treatment of cancer is carfilzomib (Kyprolis), a selectiveproteasome inhibitor. By way of non-limiting examples, immunotherapeuticagents useful in the invention include interleukin 2 (IL2), andinterferon alpha (IFNα). One non-limiting example of a suitabletherapeutic antibody that may be used is bevacizumab (Avastin).

EXAMPLES Example 1. Disruptor-1 Trial: Study of BNC105P in Combinationwith Everolimus for Metastatic Clear Cell Renal Cell Carcinoma

The objective of the study was to determine the response rate ofpatients administered BNC105P (vascular disrupting agent) in combinationwith everolimus (mTOR inhibitor) in patients who had progressed fromprior tyrosine kinase inhibitor therapy. In addition, the presentinventors determined the improvement in 6-months progression freesurvival (PFS) with the addition of BNC105P to everolimus (Afinitor). Acontrol group of patients received everolimus alone.

The medical histories of patients were obtained prior to commencement ofthe study. Patients selected for the study exhibited a KarnofskyPerformance Score of ≥70, had metastatic or locally advanced inoperablerenal cell carcinoma, had progressive disease after 1-2 prior treatmentswith VEGF-directed tyrosine kinase inhibitors, had no active brainmetastases, and good bone marrow, liver and kidney function.

Patients in the BNC105P in combination with everolimus study group (the“combination arm”) were administered 10 mg oral everolimus daily. Ondays 1 and 8 of the 21 day cycle period, the patients were administered16 mg/m² BNC105P, which had been determined to be the maximum tolerateddose, by intravenous infusion over a 10 minute period (FIG. 1).Treatment was continued until disease progressed, intolerable toxicitybecame apparent, or until consent was withdrawn. A control group ofpatients were administered with everolimus alone.

All patients in the study were followed for disease progression andsurvival. Prior to commencing treatment, patients enrolled in the studywere subject to disease assessment including: CT of chest, abdomen andpelvis; CT of the head or brain MRI; bone scan; echocardiography (ECHO)or multiple gated acquisition scan (MUGA). At every 3 cycles oftreatment, CT scans of chest, abdomen and pelvis, and bone scans, wereperformed to determine disease progression.

Example 2. Disease Evaluation and Response Criteria

The criteria for disease evaluation included:

Measurable disease:—the presence of at least one measurable lesion. Ifthe measurable disease is restricted to a solitary lesion, itsneoplastic nature should be confirmed by cytology/histology.

Measurable lesions:—lesions that can be accurately measured in at leastone dimension with longest diameter (LD)>20 mm using conventionaltechniques or >10 mm with spiral CT scan.

Non-measurable lesions:—all other lesions, including small lesions(longest diameter <20 mm with conventional techniques or <10 mm withspiral CT scan), i.e., bone lesions, leptomeningeal disease, ascites,pleural/pericardial effusion, inflammatory breast disease, lymphangitiscutis/pulmonis, cystic lesions, and also abdominal masses that are notconfirmed and followed by imaging techniques.

Baseline documentation of “target” and “non-target” lesions:—allmeasurable lesions up to a maximum of five lesions per organ and 10lesions in total, representative of all involved organs should beidentified as target lesions and recorded and measured at baseline.Target lesions should be selected on the basis of their LD. The baselinesum LD will be used as reference by which to characterize the objectivetumor. All other lesions (or sites of disease) should be identified asnon-target lesions and should also be recorded at baseline. Measurementsof these lesions are not required, but the presence or absence of eachshould be noted throughout follow-up.

The response criteria for the evaluation of target lesions were asfollows:

Complete Response (CR): Disappearance of all target lesions

Partial Response (PR): At least a 30% decrease in the sum of the LD oftarget lesions, taking as reference the baseline sum LD

Progressive Disease (PD): At least a 20% increase in the sum of the LDof target lesions, taking as reference the smallest sum LD recordedsince the treatment started or the appearance of one or more new lesions

Stable Disease (SD): Neither sufficient shrinkage to qualify for PR norsufficient increase to qualify for PD, taking as reference the smallestsum LD since the treatment started

The response criteria for the evaluation of non-target lesions were asfollows:

Complete Response (CR) Disappearance of all non-target lesions andnormalization of tumor marker level

Incomplete Response/Stable Disease (SD): Persistence of one or morenon-target lesion(s) or/and maintenance of tumor marker level above thenormal limits

Progressive Disease (PD): Appearance of one or more new lesions and/orunequivocal progression of existing non-target lesions

Time to disease progression was determined as a measurement from thestart of the treatment until the criteria for disease progression is met(or death occurs), taking as reference the smallest measurementsrecorded since the treatment started. The time to progression, inpatients with documented disease progression at their first diseaseevaluation, were considered the time between initiation of therapy andthe date of first documentation of disease progression.

Example 3. Biological Sample Processing

Human EDTA plasma samples were taken from patients to quantitativelymeasure the concentration of 56 analytes from the Human CustomMAP atMyriad RBM (MRBM). All samples were received, processed and storedaccording to established MRB procedures. The samples were thawed at roomtemperature, vortexed, centrifuged for clarification and adequate volumewas removed for Human CustomMAP analysis and plated into a mastermicrotiter plate.

Multi-Analyte Profile (MAP) Technology

MRBM has developed species-specific MAPs based on Luminex technologywith capability to simultaneously measure multiple biochemical markersin a very small sample volume. Luminex technology performs up to 100multiplexed, microsphere-based assays in a single reaction vessel bycombining optical classification schemes, biochemical assays, flowcytometry and advanced digital signal processing hardware and software.

Multiplexing is accomplished by assigning each analyte-specific assay amicrosphere set labelled with a unique fluorescence signature. To attain100 distinct microsphere signatures, two fluorescent dyes, red and farred, are mixed in various combinations using ten intensity levels ofeach dye (i.e., 10×10). Each batch or set of microspheres is encodedwith a fluorescent signature by impregnating the microspheres with oneof these dye combinations.

After the encoding process, an assay-specific capture reagent (i.e.,antigens, antibodies, receptors, peptides, enzyme substrates, etc.) isconjugated covalently to each unique set of microspheres. Covalentattachment of the capture reagent to the microspheres is achieved withstandard carbodiimide chemistry using carboxyl functional groups locatedon the surface of each 5.6 μm microsphere and primary amines within thecapture reagent. Coupling chemistry is performed on large numbers ofindividual microspheres (10⁷-10⁹ microspheres/mL) simultaneously withineach unique set, resulting in low microsphere-to-microspherevariability.

After optimizing the parameters of each assay separately, Multi-AnalyteProfiles are performed by mixing up to 100 different sets of themicrospheres in a single well of a 96- or 384-format microtiter plate. Asmall sample volume is added to the well and allowed to react with themicrospheres. The assay-specific capture reagent on each individualmicrosphere binds the analyte of interest. A cocktail of assay-specific,biotinylated detecting reagents (e.g., antigens, antibodies, ligands,etc.), is reacted with the microsphere mixture, followed by astreptavidin-labeled fluorescent “reporter” molecule (typicallyphycoerythrin). Because the microspheres are in suspension, the assaykinetics are near solution-phase. Finally, the multiplex is washed toremove unbound detecting reagents.

After washing, the mixture of microspheres is analyzed using the Luminex100/200™ instrument. Similar to a flow cytometer, the instrument useshydrodynamic focusing to pass the microspheres in single file throughtwo laser beams.

As each individual microsphere passes through the excitation beams, itis analyzed for size, encoded fluorescence signature and the amount offluorescence generated in proportion to the analyte. Microsphere size,determined by measuring the 90-degree light scatter as the microspherespass through a red diode laser (633 nm), is used to eliminatemicrosphere aggregates from the analysis. While in the red excitationbeam, the encoded red and far red dyes are excited and the resultingfluorescence signature (ratio 660 nm/720 nm) is filtered, measured usingavalanche photodiodes, and classified to a microsphere set. Since eachmicrosphere is encoded with a unique signature, the classificationidentifies the analyte being measured on that individual microsphere. Asthe microsphere passes through a green diode-pumped solid state laser(532 nm), a fluorescence “reporter” signal (580 nm) is generated inproportion to the analyte concentration, filtered and measured using aphotomultiplier tube.

Luminex Testing

Using automated pipetting on Tecan robots, an aliquot of each sample wasintroduced into one of the capture microsphere multiplexes of the HumanCustomMAP. These mixtures of sample and capture microspheres werethoroughly mixed and incubated at room temperature for 1 hour.Multiplexed cocktails of biotinylated, reporter antibodies for eachmultiplex were then added robotically and after thorough mixing, wereincubated for an additional hour at room temperature.

Multiplexes were developed using an excess of streptavidin-phycoerythrinsolution which was thoroughly mixed into each multiplex and incubatedfor 1 hour at room temperature. The volume of each multiplexed reactionwas reduced by vacuum filtration and the volume increased by dilutioninto matrix buffer for analysis. Analysis was performed in a Luminexinstrument and the resulting data stream was interpreted using dataanalysis software.

Assays were run in high density multiplexed panels and the LeastDetectable Dose (LDD) was determined as the mean +3 standard deviationsof 20 blank readings. The LLOQ is determined by the concentration of ananalyte where the measurement of analyte demonstrates a coefficient ofvariation (CV) of 30%. It represents the lowest concentration of analytethat can be measured with a precision better than or equal to 30%.Appropriate dilutions were made to ensure a quantitative measurementwithin the limits of the assay.

An eight (n=8) point standard curve (S1-S8) was used to obtainquantitative measurements for each sample. Quality Controls (QC's) (n=3;C1-C3) were run in duplicate along different points of the curve toensure both accuracy and precision for each analyte.

Chemicals and Solutions

Streptavidin-Phycoerythrin was purchased from Molecular Probes™. Allbuffers, reagents, capture microsphere multiplexes of the HumanCustomMAP, multiplexed cocktails of biotinylated reporter antibodies,and multiplexed standards and controls were prepared by MRBM.

Example 4: Methods of Evaluation and Statistical Analysis

Data acquisition, analysis and reporting (MRBM Plate Viewer) wereperformed in real-time on all microsphere sets included in the MAP. Aminimum of twenty individual microspheres from each unique set wereanalyzed and the median value of the analyte-specific, or “reporter,”fluorescence were logged. Using calibrators and assay controls,specific, sensitive, and quantitative results were achieved withprecision enhanced by the analysis of at least twenty microspheres perdata point.

All safety analyses were performed using the Intent-To-Treat (ITT)population. The standard summary statistics for continuous variableswere: mean, standard deviation, median, quartiles, maximum and minimum.The standard summary statistics for discrete values were: count andproportion. Progression free survival (PFS) was defined as date on studyto date of disease progression or death, and overall survival ofpatients was summarized by Kaplan-Meier methods performed among the ITTpopulation.

The 6-month PFS and response rates among evaluable patients weresummarized by proportions together with 95% confidence intervals. Countsand proportions by toxicity were presented with exact binomial 95%confidence intervals for the following (ITT population).

The biomarker levels at each time and the change in biomarker levelswere examined. The mean change from pre- to post-infusion was calculatedas the mean of C1D1 pre-infusion minus C1D1 post-infusion. Thus, apositive mean change from pre- to post-infusion indicates a reduction inthe biomarker over infusion time.

Example 5. Results

There were 56 biomarkers. For 10 of the biomarkers, all samples valueswere below the limit of detection of the assay so no analyses werepossible. An additional 2 biomarkers had many values below thedetectable limit of the assay so the analysis with progression by 6months was unable to be performed. The present inventors correlatedbiomarker changes induced by BNC105P infusion with progression freesurvival. The dosing schedule and biomarker sampling time points areprovided in FIG. 1.

Biomarkers were analysed to determine those which exhibited astatistically significant association between biomarker plasmaconcentration change and progression free survival. BNC105P-inducedbiomarker plasma concentration changes were detectable after the initialadministration of BNC105P on Cycle 1/Day 1 (C1D1), both at the 1 hourpost-administration sampling time and at 3 hours post administrationsampling time.

The five biological markers for which a change in concentrationpre-administration versus post-administration was detected were SerumAmyloid P-Component (SAP), Sex Hormone-Binding Globulin (SHBG),Myoglobin (MB), Matrix Metalloproteinase-9 (MMP-9), and Stem Cell Factor(SCF). Analysis of the change in biomarker levels is shown in Table 2.

TABLE 2 BNC105P induced changes in Plasma Biomarkers correlate with 6month Progression Free Survival Biomarker change Median Ratio RangePost/Baseline Correlation with associated with Ratio PFS (P value)better PFS SAP 0.95 0.0184 ≤0.95 SHBG 0.92 0.0063 ≤0.92 Myoglobin 0.9350.0011 ≤0.935 MMP-9 1.060 0.042 >1.060 SCF 1.0 0.0291 >1

The ratio of post-versus pre-administration levels of the biologicalmarkers listed in Table 1 are shown in FIGS. 2A, 3A, 4A, 5A and 6A.Samples were taken from patients at both 1 hour and 3 hours postadministration with BNC105P and exhibited consistent changes in markerlevel at both time points. The median level of the biological markers ateach time point in these Figures is indicated with a solid line.

FIGS. 2B, 3B, 4B, 5B and 6B demonstrate that patients separated into twogroups based on the median of the ratio of biomarker post administrationwith BNC105P versus the baseline (pre-administration) level of thebiomarker. From these Figures, it can be seen that the patientpopulation that exhibited a decrease in levels of SAP, SHBG or Myoglobinfollowing administration with BNC105P had greater progression freesurvival compared to rest of the patient population. In contrast, thepatient population that had an increase in the level of MMP-9 or SCFfollowing administration with BNC105P had greater progression freesurvival compared to the rest of the patient population.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the scope of theinvention as broadly described. The present embodiments are, therefore,to be considered in all respects as illustrative and not restrictive.

All publications discussed and/or referenced herein are incorporatedherein in their entirety.

The present application claims priority from AU 2014902541 filed 2 Jul.2014, the entire contents of which are incorporated herein by reference.

Any discussion of documents, acts, materials, devices, articles or thelike which has been included in the present specification is solely forthe purpose of providing a context for the present invention. It is notto be taken as an admission that any or all of these matters form partof the prior art base or were common general knowledge in the fieldrelevant to the present invention as it existed before the priority dateof each claim of this application.

REFERENCES

-   Gershagen et al. (1989) Nucleic Acids Res, 17:9245-9258-   Mantzouranis et al. (1985) J Biol Chem, 260:7752-7756-   Metzker (2010) Nat Rev Genet, 11(1):31-46

The invention claimed is:
 1. A method of treating cancer in a patient,the method comprising: identifying a patient having a level of abiological marker prior to administration with a vascular disruptingagent which is different to the level of the biological marker followingadministration with the vascular disrupting agent, wherein thebiological marker is selected from Serum Amyloid P (SAP), SexHormone-Binding Globulin (SHBG), Myoglobin, and/or Stem Cell Factor(SCF), and administering the vascular disrupting agent to the patient,wherein a level of a biological marker prior to administration with thevascular disrupting agent that is different to the level of thebiological marker following administration with the vascular disruptingagent is indicative of the patient responding to treatment with thevascular disrupting agent, and wherein the vascular disrupting agent isa compound of formula (I) or a salt, solvate or prodrug thereof

wherein: X represents O, S, SO, SO₂, Se, SeO, SeO₂ or NR where R isselected from H, O, optionally substituted acyl, optionally substitutedalkenyl, optionally substituted alkyl, optionally substituted aryl,optionally substituted cycloalkenyl, optionally substituted cycloalkyl,optionally substituted heteroaryl, optionally substituted heterocyclyl,and optionally substituted sulfonyl; R^(1A) and R^(1B) eachindependently represents H, carboxy, cyano, dihalomethoxy, halogen,hydroxy, nitro, pentahaloethyl, phosphorylamino, phosphono, phosphinyl,sulfo, trihaloethenyl, trihalomethanethio, trihalomethoxy,trihalomethyl, optionally substituted acyl, optionally substitutedacylamino, optionally substituted acylimino, optionally substitutedacyliminoxy, optionally substituted acyloxy, optionally substitutedarylalkyl, optionally substituted arylalkoxy, optionally substitutedalkenyl, optionally substituted alkenyloxy, optionally substitutedalkoxy, optionally substituted alkyl, optionally substituted alkynyl,optionally substituted alkynyloxy, optionally substituted amino,optionally substituted aminoacyl, optionally substituted aminoacyloxy,optionally substituted aminosulfonyl, optionally substitutedaminothioacyl, optionally substituted aryl, optionally substitutedaryloxy, optionally substituted cycloalkenyl, optionally substitutedcycloalkyl, optionally substituted heteroaryl, optionally substitutedheterocyclyl, optionally substituted oxyacyl, optionally substitutedoxyacylamino, optionally substituted oxyacyloxy, optionally substitutedoxyacylimino, optionally substituted oxysulfinylamino, optionallysubstituted oxysulfonylamino, optionally substituted oxythioacyl,optionally substituted oxythioacyloxy, optionally substituted sulfinyl,optionally substituted sulfinylamino, optionally substituted sulfonyl,optionally substituted sulphonylamino, optionally substituted thio,optionally substituted thioacyl, optionally substituted thioacylamino,or R^(1A) and R^(1B) together form an optionally substituted aryl,optionally substituted heterocyclyl, optionally substituted heteroaryl,optionally substituted cycloalkyl, or optionally substitutedcycloalkenyl; R^(1C) represents C₁₋₃ alkoxy, C₁₋₃ alkylthio, C₁₋₃alkylamino, or C₁₋₃ dialkylamino; R^(1D) represents hydroxy or amino; Lrepresents C═O, O, S, SO, SO₂, Se, SeO, SeO₂, C═NZ′, or NR′ where Z′ isH, optionally substituted alkyl, optionally substituted aryl oroptionally substituted amino; and where R′ is selected from H, O,optionally substituted acyl, optionally substituted alkenyl, optionallysubstituted alkyl, optionally substituted aryl, optionally substitutedcycloalkenyl, optionally substituted cycloalkyl, optionally substitutedheteroaryl, optionally substituted heterocyclyl, or optionallysubstituted sulfonyl; R^(2A)-R^(2E) each independently represents H,carboxy, cyano, dihalomethoxy, halogen, hydroxy, nitro, pentahaloethyl,phosphorylamino, phosphono, phosphinyl, sulfo, trihaloethenyl,trihalomethanethio, trihalomethoxy, trihalomethyl, optionallysubstituted acyl, optionally substituted acylamino, optionallysubstituted acylimino, optionally substituted acyliminoxy, optionallysubstituted acyloxy, optionally substituted arylalkyl, optionallysubstituted arylalkoxy, optionally substituted alkenyl, optionallysubstituted alkenyloxy, optionally substituted alkoxy, optionallysubstituted alkyl, optionally substituted alkynyl, optionallysubstituted alkynyloxy, optionally substituted amino, optionallysubstituted aminoacyl, optionally substituted aminoacyloxy, optionallysubstituted aminosulfonyl, optionally substituted aminothioacyl,optionally substituted aryl, optionally substituted aryloxy, optionallysubstituted cycloalkenyl, optionally substituted cycloalkyl, optionallysubstituted heteroaryl, optionally substituted heterocyclyl, optionallysubstituted oxyacyl, optionally substituted oxyacylamino, optionallysubstituted oxyacylimino, optionally substituted oxyacyloxy, optionallysubstituted oxysulfinylamino, optionally substituted oxysulfonylamino,optionally substituted oxythioacyl, optionally substitutedoxythioacyloxy, optionally substituted sulfinyl, optionally substitutedsulfinylamino, optionally substituted sulfonyl, optionally substitutedsulphonylamino, optionally substituted thio, optionally substitutedthioacyl, optionally substituted thioacylamino, or optionallysubstituted thioacyloxy; or any of R^(2A) and R^(2B), R^(2B) and R^(2C),R^(2C) and R^(2D), and R^(2D) and R^(2E), together form an optionallysubstituted aryl, optionally substituted heterocyclyl, optionallysubstituted heteroaryl, optionally substituted cycloalkyl, or optionallysubstituted cycloalkenyl; and Q represents H, CN, halogen,trialkylsilyl, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substituted acyl,optionally substituted oxyacyl, optionally substituted acylamino,optionally substituted aminoacylamino, OR″, SR″ or NR″R″, where each R″independently represents, H, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted aryl, optionally substituted heteroaryl, optionallysubstituted heterocyclyl, optionally substituted acyl and optionallysubstituted oxyacyl, or NR′″NR′″, where each R′″ independentlyrepresents H, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substituted aryl andoptionally substituted heteroaryl, and wherein the method comprisesdetermining a level of the biological marker in a sample obtained fromthe patient following administration with the vascular disrupting agent,wherein the sample is a tumor, blood, serum or plasma.
 2. The method ofclaim 1, wherein the wherein the level of the biological marker isdetermined by measuring the level of biological marker polypeptide. 3.The method of claim 1, wherein the biological marker is selected fromSAP, SHBG, and/or Myoglobin and the level of the biological marker islower following administration with the vascular disrupting agent whencompared to the level of the biological marker prior to administrationwith the vascular disrupting agent.
 4. The method of claim 1, whereinthe biological marker is SCF and the level of the biological marker ishigher following administration with the vascular disrupting agent whencompared to the level of the biological marker prior to administrationwith the vascular disrupting agent.
 5. The method of claim 1, whereinthe vascular disrupting agent is selected from2-methyl-7-hydroxy-3-(3,4,5-trimethoxybenzoyl)-6-methoxybenzofuran anddisodium[6-methoxy-2-methyl-3-(3,4,5-trimethoxybenzoyl)-1-benzofuran-7-yl]phosphate.
 6. The method of claim 1, wherein the patient is a renalcancer patient.
 7. The method of claim 1, comprising administering afurther therapeutic agent and/or tumor irradiation to the patient. 8.The method of claim 7, wherein the further therapeutic agent is selectedfrom a chemotherapeutic, an antibody and/or an immunotherapeutic.
 9. Themethod of claim 7, wherein the further therapeutic agent is selectedfrom an mTOR inhibitor, tyrosine kinase inhibitor and/or a VEGFinhibitor.